Anti-CD70 antibody drug conjugates

ABSTRACT

This invention relates to anti-CD70 antibodies and antibody drug conjugates comprising at least one non-naturally-encoded amino acid. Disclosed herein are αCD70 antibodies with one or more non-naturally encoded amino acids and further disclosed are antibody drug conjugates wherein the αCD70 antibodies of the invention are conjugated to one or more toxins. Further disclosed are methods for using such non-natural amino acid antibody drug conjugates, including therapeutic, diagnostic, and other biotechnology uses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is national phase entry in the United States under 35U.S.C. § 371 of International Patent Application No. PCT/US13/46669filed on Jun. 19, 2013, which is incorporated by reference herein in itsentirety and claims priority to and the benefit of U.S. ProvisionalApplication Ser. No. 61/661,782 filed on Jun. 19, 2012, thespecification and contents of which are incorporated by reference in itsentirety for all purposes.

FIELD OF THE INVENTION

This invention relates to anti-CD70 antibodies and antibody drugconjugates comprising at least one non-naturally-encoded amino acid.Disclosed herein are αCD70 antibodies with one or more non-naturallyencoded amino acids and further disclosed are antibody drug conjugateswherein the αCD70 antibodies of the invention are conjugated to one ormore toxins. Further disclosed are methods for using such non-naturalamino acid antibody drug conjugates, including therapeutic, diagnostic,and other biotechnology uses.

BACKGROUND OF THE INVENTION

CD70 is a member of the tumor necrosis factor (TNF) family of cellmembrane-bound and secreted molecules that are expressed by a variety ofnormal and malignant cell types. The primary amino acid (AA) sequence ofCD70 predicts a transmembrane type II protein with its carboxyl terminusexposed to the outside of cells and its amino terminus found in thecytosolic side of the plasma membrane (Bowman et al., 1994, J Immunol152:1756-61; Goodwin et al., 1993, Cell 73:447-56). Human CD70 iscomposed of a 20 AA cytoplasmic domain, an 18 AA transmembrane domain,and a 155 AA extracytoplasmic domain with two potential N-linkedglycosylation sites (Bowman et al., supra; Goodwin et al., supra).Specific immunoprecipitation of radioisotope-labeled CD70-expressingcells by anti-CD70 antibodies yields polypeptides of 29 and 50 kDa(Goodwin et al., supra; Hintzen et al., 1994, J Immunol 152:1762-73).Based on its homology to TNF-alpha and TNF-beta, especially instructural strands C, D, H and I, a trimeric structure is predicted forCD70 (Petsch et al., 1995, Mol Immunol 32:761-72).

Original immunohistological studies revealed that CD70 is expressed ongerminal center B cells and rare T cells in tonsils, skin, and gut(Hintzen et al., 1994, Int Immunol 6:477-80). Subsequently, CD70 wasreported to be expressed on the cell surface of recentlyantigen-activated T and B lymphocytes, and its expression wanes afterthe removal of antigenic stimulation (Lens et al., 1996, Eur J Immunol26:2964-71; Lens et al., 1997, Immunology 90:38-45). Within the lymphoidsystem, natural killer cells (Orengo et al., 1997, Clin Exp Immunol107:608-13) and mouse mature peripheral dendritic cells (Akiba et al.,2000, J Exp Med 191:375-80) also express CD70. In non-lymphoid lineages,CD70 has been detected on thymic medullar epithelial cells (Hintzen etal., 1994, supra; Hishima et al., 2000, Am J Surg Pathol 24:742-46).

In addition to expression on normal cells, CD70 expression has beenreported in different types of cancers including lymphomas, carcinomas,and tumors of neural origin. In malignant B cells, 71% of diffuse largeB-cell lymphomas, 33% of follicle center lymphomas, 25% of mantlelymphomas, and 50% of B-CLL have been reported to express CD70 (Lens etal., 1999, Br J Haematol 106:491-503). CD70 is frequently expressedtogether with other lymphoid activation markers on the malignant Hodgkinand Reed-Sternberg cells of Hodgkin's disease (Gruss and Kadin, 1996,Bailieres Clin Haematol 9:417-46). One report demonstrates CD70expression on 88% (7 of 8 cases) of thymic carcinomas and 20% (1 of 5cases) of atypical thymomas (Hishima et al., 2000, supra). The secondtype of carcinoma on to which CD70 has been detected is nasopharyngealcarcinoma. One study reports the presence of CD70 on 80% (16 of 20cases) of snap-frozen tumor biopsies obtained from undifferentiatednasopharyngeal carcinomas (Agathanggelou et al., 1995, Am J Path147:1152-60). CD70 has also been detected on brain tumor cells,especially glioma cell lines, solid human gliomas, and meningiomas(Held-Feindt and Mentlein, 2002, Int J Cancer 98:352-56; Wischlusen etal., 2002, Can Res 62:2592-99).

It has been proposed that transforming viruses including theEpstein-Barr virus (EBV) and the human T leukemia virus-1 (HTLV-1) caninduce CD70 on cells such as epithelial cells that normally do notexpress CD70 (Agathanggelou et al., supra; Stein et al., 1989, OxfordUniversity Press, p. 446). Therefore, expression of CD70 on malignant Bcells could be a reflection of oncogenic transformation (Lens et al.,1999, supra). Also, since CD70 expression is induced on B cells afterantigen encounter (Maurer et al., 1990, Eur J Immunol 20:2679-84; Lenset al., 1996, supra), stable expression of CD70 might reflect prolongedantigenic stimulation. This has been postulated to occur in follicularnon-Hodgkin's lymphomas based on ongoing somatic hypermutation (Bahleret al., 1992, Proc Natl Acad Sci USA 89:6770-74; Bahler et al., 1992,Cancer Res 52:suppl. 5547S-51S).

The receptor for CD70 is CD27, a glycosylated type I transmembraneprotein of about 55 kDa (Goodwin et al., 1993, Cell 73:447-56; Hintzenet al., 1994, supra). CD70 is sometimes referred to as CD27L. CD27,which exists as a homodimer on the cell surface (Gravestein et al.,1993, Eur J Immunol 23:943-50), is a member of the TNF receptorsuperfamily as defined by cysteine-rich repeats of about 40 amino acidsin the extracellular domain (Smith et al., 1990, Science 248:1019-23;Locksley et al., 2001, Cell 104:487-501). CD27 is typically expressed bythymocytes, NK, T, and B cells (Hintzen et al., 1994, Immunol Today15:307-11; Lens et al., 1998, Semin Immunol 10:491-99). On resting Tcells, CD27 is constitutively expressed, yet antigenic triggeringfurther upregulates CD27 expression (de Jong et al., 1991, J Immunol146:2488-94; Hintzen et al., 1993, J. Immunol. 151:2426-35). Further,triggering of T cells via their T cell antigen receptor complex alone orin combination with the accessory molecule CD28 releases soluble CD27from activated T cells (Hintzen et al., 1991, J Immunol 147:29-35).Naive B cells do not express CD27, but its expression is induced and, incontrast to CD70, sustained after antigenic triggering of B cells(Jacquot S et al., 1997 J Immunol 159:2652-57; Kobata T et al., 1995,Proc Natl Acad Sci USA 92:11249-53).

In marked contrast to the restricted expression of CD27 and CD70 innormal B lineage cells, both CD27 and CD70 are frequently co-expressedin many B cell non-Hodgkin's lymphomas and leukemias. This couldpotentially lead to functional CD27-CD70 interactions on these cells inthe form of an autocrine loop, resulting in CD27 signaling and inCD70-induced proliferation, thereby providing a growth advantage tomalignant cells (Lens et al., 1999, supra).

The available data supports a model in which ligation of CD27 onactivated lymphocytes by CD70 delivers signals to the CD27-expressingcells, including co-stimulatory signals in T, B, and NK cells. (See,e.g., Goodwin et al., supra; Hintzen et al., 1995, J Immunol154:2612-23; Oshina et al., 1998, Int Immunol 10:517-26; Smith et al.,supra; Van Lier et al., 1987, J Immunol 139:1589-96; Gravestein et al.,1995, Int Immunol 7:551-7; Tesselaar et al., 1997, J Immunol159:4959-65; Jacquot et al., supra; Agematsu et al., 1998, Blood91:173-80; Kobata et al., supra; Agematsu et al., 1997, Eur J Immunol27:2073-79; Sugita et al., 1992, J Immunol 149:1199-1203; Orengo et al.,1997, Clin Exp Immunol 107:608-13). Antibodies against both murine andhuman CD70 have been demonstrated to inhibit such activities, presumablyby blocking the CD70/CD27 interaction (Hintzen et al., 1994, supra;Hintzen et al., 1995, supra; Oshima et al., supra).

Limited information is available on the modulation of cellular functionsthrough CD70 signaling upon CD70/CD27 interaction, i.e., ‘reversesignaling’. Some CD70 antibodies have the ability to enhance T cellproliferation when they are presented to CD70-expressing T cells eithercross-linked with a secondary antibody or immobilized on tissue cultureplates (Bowman et al., 1994, J Immunol 152:1756-61; Brugnoni, 1997,Immunol Lett 55:99-104). Such ‘reverse signaling’ has also beendescribed in a subset of B chronic lymphocytic leukemia (B-CLL) cells,and CD70 can function as a receptor to transduce signals to facilitateproliferation of PMA-stimulated purified B-CLL cells (Lens et al., 1999,supra). These observations suggest situations in which engagement ofCD27 and CD70 can result in the delivery of agonistic signals to boththe CD27 and CD70 expressing cells.

The role of CD70/CD27 co-stimulation in cell-mediated autoimmunediseases has been investigated in a model of experimental autoimmuneencephalomyelitis (EAE) (Nakajima et al., 2000, J Neuroimmunol109:188-96). In vivo administration of a particular anti-mouse CD70 mAb(clone FR-70) markedly suppressed the onset of EAE by inhibitingantigen-induced TNF-alpha production without affecting T cell priming,Ig production or T_(H1)/T_(H2) cell balance. However, such treatment hadlittle efficacy in established disease. It has been reported thatexpression of CD70 on T cells was enhanced by TNF-alpha and IL-12 anddown regulated by IL₄ (Lens et al., 1998, supra). Thus, the CD70/CD27mediated T cell-T cell interactions may play a role in enhancingT_(H1)-mediated immune responses rather than T_(H2)-mediated responses.Supporting this hypothesis, the anti-mouse CD70 mAb FR-70 is alsoeffective in inhibiting T_(H1)-mediated collagen-induced arthritis(Nakajima et al., 2000, supra). In contrast, the same anti-mouse CD70mAb did not show any efficacy in modulating lupus in NZB/NZW F1 mice andexperimental Leishmania major infection in susceptible BALB/c mice, bothof which are predominantly T_(H2)-mediated autoimmune response (Nakajimaet al., 1997, J Immunol 158, 1466-72; Akiba et al., 2000, J Exp Med191:375-380).

The role of CD70 has not yet been investigated in acute graft versushost disease (aGVHD), another T_(H1)-mediated immune response. GVHD is amajor and often lethal consequence of allogeneic bone marrowtransplantation (BMT) therapy that occurs when histocompatibilityantigen differences between the BM donor and the recipient of thetransplant are present (den Haan et al., 1995, Science 268:1476). GVHDis caused by mature T cells present in the transplanted marrow, as wellas other minor cell populations (Giralt and Champlin, 1994, Blood84:3603). It is noteworthy that CD70 has been detected in vivo on CD4+cells in conditions characterized by allogeneic reaction, as in cases ofmaternal T cell engraftment in severe combined immune deficiencypatients (Brugnoni et al., Immunol Lett 55:99-104). Prophylaxis of GVHDis achieved by pan-T cell immunosuppressive agents such as cyclosporine,corticosteroids, or methotrexate. In addition to the lack ofspecificity, these agents are also associated with significant adverseside effects. To limit these undesirable effects and the disruption ofnormal T cell functions, other therapeutic interventions based onselective targeting of T cells directly participating inallo-recognition and graft rejection are much needed.

CD70 is a potentially useful target for antibody-directed immunotherapy.As indicated supra, CD70 has a restricted expression pattern in normalcells: CD70 expression is mostly restricted to recentlyantigen-activated T and B cells under physiological conditions, and itsexpression is down-regulated when antigenic stimulation ceases. The keyrole of CD70 is believed to be facilitating plasma cell differentiationand contributing to the generation and maintenance of long-term T cellmemory. Further, evidence from animal models suggests that unregulatedCD70/CD27 interaction may contribute to immunological disorders, and, inhumans, experimental data have also pointed towards potential abnormalregulation of the CD70/CD27 pathway in T_(H1)-mediated immune disorderssuch as, e.g., rheumatoid arthritis, psoriasis, and multiple sclerosis.It is of particular interest that CD70 is expressed on a variety oftransformed cells including lymphoma B cells, Hodgkin and Reed-Sternbergcells, malignant cells of neural origin, and a number of carcinomas.

Several groups have demonstrated the inhibitory effect of anti-CD70 mAbin both in vitro models of lymphocyte activation and animal models ofT_(H1)-mediated responses. The focus has been on the use of antibodiesto block the CD70/CD27 co-stimulation pathway to achieve therapeuticefficacy. However, one main shortcoming of such an approach is the largenumber of signaling receptors, e.g., the CD28/CD80/CD86 co-stimulatorypathway, known to participate in immunological diseases. Consequently,blocking one specific signaling pathway may only have minimal impact ondisease development. This is supported by the observations thatanti-CD70 mAb can only partially inhibit in vitro T cell activationinduced by allogeneic stimulator cells (Hintzen et al., 1995, supra) andan anti-CD70 mAb showed no therapeutic efficacy in EAE once the diseaseis established (Nakajima et al., 2000, supra).

Thus, there is a need in the art for developing an approach fordepleting or inhibiting the growth of CD70-expressing cells involved incancers and/or immunological diseases by means other than or in additionto blocking the CD70/CD27 interaction. As CD70 is expressed on thesurface of mature antigen presenting dendritic cells, activated T cells,and activated B cells, agents that can target and inhibit or depleteCD70⁺ cells may prove to be effective in the removal of antigenpresenting cells presenting autoantigens and offending autoreactiveactivated T or B cells, as wells as CD70-expressing tumor cells.

Approaches that have been used for increasing the therapeutic efficacyof antibodies are radiolabeling and combination with chemotherapy;however, these approaches include associated with undesirable sideeffects. For example, isotope therapy is associated withmyelosuppression (Witzig, 2001, Cancer Chemother Pharmacol 48 (Suppl1):S91-5), and combining therapy with antibodies and chemotherapeuticsis associated with immunosuppression. Further, isotopically labeledsubstances are difficult to produce, and patients often experiencerelapse after initial treatment with isotopically labeled substances.

Accordingly, there is a need for anti-CD70 ADCs that are constructed insuch a manner so as to be capable exerting a clinically usefulcytotoxic, cytostatic, or immunosuppressive effect on CD70-expressingcells, particularly without exerting undesirable effects onnon-CD70-expressing cells. Such compounds would be useful therapeuticagents against cancers that express CD70 or immune disorders that aremediated by CD70-expressing cells. More recently, MAbs such as 2H5 havebeen identified. Anti-CD70 antibody-drug conjugates that can be utilizedfor imaging, diagnostic and/or therapeutic uses are therefore needed.The present invention provides such antibody-drug conjugates for use inimmunology and oncology.

SUMMARY OF THE INVENTION

Disclosed herein are anti-CD70 antibodies linked to toxic moietiesthrough one or more non-natural amino acids with one or more linker(s),and methods for making such non-natural amino acids and polypeptides.

Some embodiments of the present invention provide an anti-CD70 antibodycomprising one or more non-naturally encoded amino acids. In someembodiments, the anti-CD70 antibody comprises one non-naturally encodedamion acid. In some embodiments, the anti-CD70 antibody comprises twonon-naturally encoded amino acids. In some embodiments, the anti-CD70antibody comprises two or more non-naturally encoded amino acids. Insome embodiments, the anti-CD70 antibody comprises 3, 4, 5, or 6 morenon-naturally encoded amino acids. or more post-translationalmodifications. In some embodiments, the anti-CD70 antibody comprises theheavy chain sequence given in SEQ ID NO. 1. In some embodiments of thepresent invention, the anti-CD70 antibody comprises SEQ ID NO.1 and 2.In some embodiments, the anti-CD70 antibody is linked to a linker,polymer, or biologically active molecule. In some embodiments, theanti-CD70 antibody is linked to a linker, polymer, or biologicallyactive molecule via a non-naturally encoded amino acid in the anti-CD70antibody.

Some embodiments of the present invention describe a toxic moiety, orsalt thereof, comprising Formula (I):

wherein:

-   -   Z has the structure of:

-   -   -   R₅ is H, COR₈, C₁-C₆alkyl, or thiazole;            -   R⁸ is OH or —NH-(alkylene-O)_(n)—NH₂;        -   R₆ is OH or H;        -   Ar is phenyl or pyridine;

    -   R₇ is C₁-C₆alkyl or hydrogen;

    -   Y is selected from the group consisting of an hydroxylamine,        methyl, aldehyde, protected aldehyde, ketone, protected ketone,        thioester, ester, dicarbonyl, hydrazine, amidine, imine,        diamine, azide, keto-amine, keto-alkyne, alkyne, cycloalkyne,        and ene-dione;

    -   L is a linker selected from the group consisting of -alkylene-,        -alkylene-C(O)—, -(alkylene-O)_(n)-alkylene-,        -(alkylene-O)_(n)-alkylene-C(O)—,        -(alkylene-O)_(n)—(CH₂)_(n′)—NHC(O)CH₂)_(n″)—C(Me)₂-S—S—(CH₂)_(n′″)—NHC(O)-(alkylene-O)_(n″″)-alkylene-,        -(alkylene-O)_(n)-alkylene-W—, -alkylene-C(O)—W—,        -(alkylene-O)_(n)-alkylene-U-alkylene-C(O)—, and        -(alkylene-O)_(n)-alkylene-U-alkylene-;        -   W has the structure of:

-   -   -   U has the structure of:

-   -   -   or L is absent, Y is methyl, R₅ is COR₈, and R₈ is            —NH-(alkylene-O)_(n)—NH₂; and

    -   each n, n′, n″, n′″ and n″″ are independently integers greater        than or equal to one.

In some embodiments, R₅ is thiazole. In other embodiments, R₆ is H. Incertain embodiments, Ar is phenyl. In further or additional embodiments,R₇ is methyl. In some embodiments, n is an integer from 0 to 20, 0 to 10or 0 to 5.

In some embodiments, a compound is described comprising Formula (II):

In certain embodiments, L is -(alkylene-O)_(n)-alkylene-. In specificembodiments, each alkylene is —CH₂CH₂—, n is equal to 3, and R₇ ismethyl. In other embodiments, L is -alkylene-. In specific embodiments,each alkylene is —CH₂CH₂— and R₇ is methyl or hydrogen. In certainembodiments, L is -(alkylene-O)_(n)-alkylene-C(O)—. In certain specificembodiments, each alkylene is —CH₂CH₂—, n is equal to 4, and R₇ ismethyl. In further or alternative embodiments, L is-(alkylene-O)_(n)—(CH₂)_(n′)—NHC(O)—(CH₂)_(n″)—C(Me)₂-S—S—(CH₂)_(n′″)—NHC(O)-(alkylene-O)_(n″″)-alkylene-.In specific embodiments, each alkylene is —CH₂CH₂—, n is equal to 1, n′is equal to 2, n″ is equal to 1, n′″ is equal to 2, n″″ is equal to 4,and R₇ is methyl.

In some embodiments, Y is azide. In other embodiments, Y is cyclooctyne.In specific embodiments, the cyclooctyne has a structure of:

-   -   each R₁₉ is independently selected from the group consisting of        C₁-C₆ alkyl, C₁-C₆ alkoxy, ester, ether, thioether, aminoalkyl,        halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl        halide, alkyl amine, alkyl sulfonic acid, alkyl nitro,        thioester, sulfonyl ester, halosulfonyl, nitrile, alkyl nitrile,        and nitro; and q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.

Some embodiments of the present invention describe a compound, or saltthereof, comprising Formula (III), (IV), (V) or (VI):

wherein:

-   -   Z has the structure of:

-   -   -   R₅ is H, COR₈, C₁-C₆alkyl, or thiazole;            -   R₈ is OH;        -   R₆ is OH or H;        -   Ar is phenyl or pyridine;

    -   R₇ is C₁-C₆alkyl or hydrogen;

    -   Y and V are each selected from the group consisting of an        hydroxylamine, methyl, aldehyde, protected aldehyde, ketone,        protected ketone, thioester, ester, dicarbonyl, hydrazine,        amidine, imine, diamine, azide, keto-amine, keto-alkyne, alkyne,        cycloalkyne, and ene-dione;

    -   L₁, L₂, L₃, and L₄ are each linkers independently selected from        the group consisting of a bond, -alkylene-,        -(alkylene-O)_(n)-alkylene-J-,        -alkylene′-J-(alkylene-O)_(n)-alkylene-,        -J-(alkylene-O)_(n)-alkylene-,        -(alkylene-O)_(n)-alkylene-J-(alkylene-O)_(n)′-alkylene-J′-,        -(alkylene-O)_(n)-alkylene-J-alkylene′-, —W—, -alkylene-W—,        alkylene′-J-(alkylene-NMe)_(n)-alkylene-W—,        -J-(alkylene-NMe)_(n)-alkylene-W—,        -J-alkylene-NMe-alkylene′-NMe-alkylene″-W—, and        -alkylene-J-alkylene′-NMe-alkylene″-NMe-alkylene′″-W—;        -   W has the structure of:

-   -   -   each J and J′ independently have the structure of

-   -   -   each n and n′ are independently integers greater than or            equal to one.

In certain embodiments, a compound is described comprising Formula

In certain embodiments, L₁ is -(alkylene-O)_(n)-alkylene-J-, L₂ is-alkylene′-J′-(alkylene-O)_(n)′-alkylene-, L₃ is-J″-(alkylene-O)_(n)″-alkylene-, alkylene is —CH₂CH₂—, alkylene′ is—(CH₂)₄—, n is 1, n′ and n″ are 3, J has the structure of

J′ and J″ have the structure of

and R₇ is methyl. In other embodiments, L₁ is-J-(alkylene-O)_(n)-alkylene-, L₂ is-(alkylene-O)_(n′)-alkylene-J′-alkylene′-, L₃ is-(alkylene-O)_(n″)-alkylene-J″-, alkylene is —CH₂CH₂—, alkylene′ is—(CH₂)₄—, n is 1, n′ and n″ are 4, and J, J′ and J″ have the structureof

In some embodiments, Y is azide. In other embodiments, Y is cyclooctyne.In specific embodiments, the cyclooctyne has a structure of:

-   -   each R₁₉ is independently selected from the group consisting of        C₁-C₆ alkyl, C₁-C₆ alkoxy, ester, ether, thioether, aminoalkyl,        halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl        halide, alkyl amine, alkyl sulfonic acid, alkyl nitro,        thioester, sulfonyl ester, halosulfonyl, nitrile, alkyl nitrile,        and nitro; and    -   q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.

Certain embodiments of the present invention describe a compoundcomprising Formula (VIII) or (IX):

wherein:

-   -   A is optional, and when present is lower alkylene, substituted        lower alkylene, lower cycloalkylene, substituted lower        cycloalkylene, lower alkenylene, substituted lower alkenylene,        alkynylene, lower heteroalkylene, substituted heteroalkylene,        lower heterocycloalkylene, substituted lower        heterocycloalkylene, arylene, substituted arylene,        heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;    -   B is optional, and when present is a linker selected from the        group consisting of lower alkylene, substituted lower alkylene,        lower alkenylene, substituted lower alkenylene, lower        heteroalkylene, substituted lower heteroalkylene, —O—,        —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or        substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or 3,        —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,        —C(O)-(alkylene or substituted alkylene)-, —C(S)—,        —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,        —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,        —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,        —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene        or substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,        —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—,        —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═,        —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ is        independently H, alkyl, or substituted alkyl;    -   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted        cycloalkyl;    -   R₁ is H, an amino protecting group, resin, at least one amino        acid, polypeptide, or polynucleotide;    -   R₂ is OH, an ester protecting group, resin, at least one amino        acid, polypeptide, or polynucleotide;    -   R₃ and R₄ are each independently H, halogen, lower alkyl, or        substituted lower alkyl, or R₃ and R₄ or two R₃ groups        optionally form a cycloalkyl or a heterocycloalkyl;    -   Z has the structure of:

-   -   -   R₅ is H, CO₂H, C₁-C₆alkyl, or thiazole;        -   R₆ is OH or H;        -   Ar is phenyl or pyridine;

    -   R₇ is C₁-C₆alkyl or hydrogen;

    -   L is a linker selected from the group consisting of -alkylene-,        -alkylene-C(O)—, -(alkylene-O)_(n)-alkylene-,        -(alkyleneO)_(n)-alkylene-C(O)—,        -(alkylene-O)_(n)—(CH₂)_(n′)—NHC(O)CH₂)_(n″)—C(Me)₂-S—S—(CH₂)_(n′″)—NHC(O)-(alkylene-O)_(n″″)-alkylene-,        -(alkylene-O)_(n)-alkylene-W—, -alkylene-C(O)—W—,        -(alkylene-O)_(n)-alkylene-U-alkylene-C(O)—, and        -(alkylene-O)_(n)-alkylene-U-alkylene-;        -   W has the structure of:

-   -   -   U has the structure of:

-   -    and        -   each n, n′, n″, n′″ and n″″ are independently integers            greater than or equal to one;    -   or an active metabolite, or a pharmaceutically acceptable        prodrug or solvate thereof.

In some embodiments, R₁ is a polypeptide. In specific embodiments, thepolypeptide is an antibody. In some embodiments, the antibody isanti-CD70. In other embodiments, R₂ is a polypeptide. In specificembodiments, the polypeptide is an anti-CD70 antibody. In certainspecific embodiments, the antibody is CD70-2H5-HA119-NCA1.

Some embodiments of the present invention describe a compound, or saltthereof, comprising Formula (X), (XI), (XII) or (XIII):

wherein:

-   -   A is optional, and when present is lower alkylene, substituted        lower alkylene, lower cycloalkylene, substituted lower        cycloalkcylene, lower alkenylene, substituted lower alkenylene,        alkynylene, lower heteroalkylene, substituted heteroalkylene,        lower heterocycloalkylene, substituted lower        heterocycloalkylene, arylene, substituted arylene,        heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;    -   B is optional, and when present is a linker selected from the        group consisting of lower alkylene, substituted lower alkylene,        lower alkenylene, substituted lower alkenylene, lower        heteroalkylene, substituted lower heteroalkylene, —O—,        —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or        substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or 3,        —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,        —C(O)-(alkylene or substituted alkylene)-, —C(S)—,        —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,        —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,        —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,        —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene        or substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,        —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—,        —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═,        —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ is        independently H, alkyl, or substituted alkyl;    -   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted        cycloalkyl;    -   R₁ is H, an amino protecting group, resin, at least one amino        acid, polypeptide, or polynucleotide;    -   R₂ is OH, an ester protecting group, resin, at least one amino        acid, polypeptide, or polynucleotide;    -   R₃ and R₄ are each independently H, halogen, lower alkyl, or        substituted lower alkyl, or R₃ and R₄ or two R₃ groups        optionally form a cycloalkyl or a heterocycloalkyl;    -   Z has the structure of:

-   -   -   R₅ is H, CO₂H, C₁-C₆alkyl, or thiazole;        -   R₆ is OH or H;        -   Ar is phenyl or pyridine;

    -   R₇ is C₁-C₆alkyl or hydrogen;

    -   L₁, L₂, L₃, and L₄ are each linkers independently selected from        the group consisting of a bond, -alkylene-,        -(alkylene-O)_(n)-alkylene-J-,        -alkylene′-J-(alkylene-O)_(n)-alkylene-,        -J-(alkylene-O)_(n)-alkylene-,        -(alkylene-O)_(n)-alkylene-J-(alkylene-O)_(n)′-alkylene-J′-,        -(alkylene-O)_(n)-alkylene-J-alkylene′-, —W—, alkylene-W—,        alkylene′-J-(alkylene-NMe)_(n)-alkylene-W—,        -J-(alkylene-NMe)_(n)-alkylene-W—,        -J-alkylene-NMe-alkylene′-NMe-alkylene″-W—, and        -alkylene-J-alkylene′-NMe-alkylene″-NMe-alkylene′″-W—;        -   W has the structure of:

-   -   -   each J and J′ independently have the structure of:

-   -    and        -   each n and n′ are independently integers greater than or            equal to one.

In some embodiments, R₁ is a polypeptide. In specific embodiments, thepolypeptide is an antibody. In certain specific embodiments, theantibody is herceptin. In other embodiments, R₂ is a polypeptide. Inspecific embodiments, the polypeptide is an antibody. In certainembodiments, the antibody is αCD70. In some embodiments, the antibody isderived from any known αCD70 antibody. In certain specific embodiments,the antibody is ARX-αCD70.

In some embodiments, provided herein is a method for derivatizing adolastatin analog comprising Formula (I), (III), (IV), (V), or (VI), themethod comprising contacting the dolastatin analog with a reagent ofFormula (XXXVII), wherein Formula (I), (III), (IV), (V), or (VI)correspond to:

wherein:

-   -   Z has the structure of:

-   -   -   R₅ is H, COR₈, C₁-C₆alkyl, or thiazole;            -   R₈ is OH or —NH-(alkylene-O)_(n)—NH₂;        -   R₆ is OH or H;        -   Ar is phenyl or pyridine;

    -   R₇ is C₁-C₆ alkyl or hydrogen;

    -   Y is NH₂—O— or methyl;

    -   L, L₁, L₂, L₃, and L₄ are each linkers selected from the group        consisting of a bond, -alkylene-, -alkylene-C(O)—,        -(alkylene-O)_(n)-alkylene-, -(alkylene-O)_(n)-alkylene-C(O)—,        -(alkylene-O)_(n)—(CH₂)—NHC(OH—CH₂)_(n″)—C(Me)₂-S—S—(CH₂)_(n′″)—NHC(O)-(alkylene-O)_(n″″)-alkylene-,        -(alkylene-O)_(n)-alkylene-W—, -alkylene-C(O)—W—,        -(alkylene-O)_(n)-alkylene-J-, -alkylene′-J-(alkylene        O)_(n)-alkylene-, -(alkylene-O)_(n)-alkylene-J-alkylene′,        -J-(alkylene-O)_(n)-alkylene-,        -(alkylene-O)_(n)-alkylene-J-(alkylene-O)_(n)′-alkylene-J′-,        —W—, -alkylene-W—, alkylene′-J-(alkylene-NMe)_(n)-alkylene-W—,        and J-(alkylene-NMe)_(n)-alkylene-W—,        -(alkylene-O)_(n)-alkylene-U-alkylene-C(O)—,        -(alkylene-O)_(n)-alkylene-U-alkylene-;        -J-alkylene-NMe-alkylene′-NMe-alkylene″-W—, and        -alkylene-J-alkylene′-NMe-alkylene″-NMe-alkylene′″W—;        -   W has the structure of:

-   -   -   U has the structure of:

-   -   -   each J and J′ independently have the structure of:

-   -   -   or L is absent, Y is methyl, R₅ is COR₈, and R₈ is            —NH-(alkylene-O)_(n)—NH₂; and        -   each n, n′, n″, n′″ and n″″ are independently integers            greater than or equal to one;

wherein Formula (XXXVII) corresponds to:

wherein:

-   -   A is optional, and when present is lower alkylene, substituted        lower alkylene, lower alkenylene, substituted lower alkenylene,        arylene, substituted arylene, heteroarylene, substituted        heteroarylene, alkarylene, substituted alkarylene, aralkylene,        or substituted aralkylene;    -   B is optional, and when present is a linker selected from the        group consisting of lower alkylene, substituted lower alkylene,        lower alkenylene, substituted lower alkenylene, —O—,        —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or        substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or 3,        —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,        —C(O)-(alkylene or substituted alkylene)-, —C(S)—,        —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,        —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,        —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,        —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene        or substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,        —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—,        —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═,        —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ is        independently H, alkyl, or substituted alkyl;        -   each R′ is independently H, alkyl, or substituted alkyl;    -   K is

-   -   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted        cycloalkyl;    -   R₁ is H, an amino protecting group, resin, at least one amino        acid, or polynucleotide;    -   R₂ is OH, an ester protecting group, resin, at least one amino        acid, or polynucleotide; and

R₃ and R₄ are each independently H, halogen, lower alkyl, or substitutedlower alkyl, or R₃ and R₄ or two R₃ groups optionally form a cycloalkylor a heterocycloalkyl.

In some embodiments, the derivatized dolastatin analog comprises atleast one oxime containing amino acid having the structure of Formula(VIII), (IX), (X), (XI), (XII), or (XIII):

In specific embodiments, the dolastatin analog is contacted with thereagent of Formula (XXXVII) in aqueous solution under mildly acidicconditions.

Certain embodiments of the present invention describe a compoundcomprising Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX):

wherein:

-   -   Z has the structure of:

-   -   -   R₅ is H, CO₂H, C₁-C₆alkyl, or thiazole;        -   R₆ is OH or H;        -   Ar is phenyl, or pyridine;

    -   R₁ is H, an amino protecting group, resin, at least one amino        acid, polypeptide, or polynucleotide;

    -   R₂ is OH, an ester protecting group, resin, at least one amino        acid, polypeptide, or polynucleotide;

    -   R₄ is H, halogen, lower alkyl, or substituted lower alkyl;

    -   R₇ is C₁-C₆alkyl or hydrogen;

    -   L, L₁, L₂, L₃, and L₄ are each linkers selected from the group        consisting of a bond, -alkylene-, -alkylene-C(O)—, -alkylene-J-,        -(alkylene-O)_(n)-alkylene-, -(alkylene-O)_(n)-alkylene-C(O)—,        -(alkylene-O)_(n)-J-, -(alkylene-O)_(n)-J-alkylene-,        -(alkylene-O)_(n)—(CH₂)_(n′)—NHC(O)—(CH₂)_(n″)—C(Me)₂-S—S—(CH₂)_(n′″)—NHC(O)-(alkylene-O)_(n″″)-alkylene-,        -(alkylene-O)_(n)-alkylene-W—, -alkylene-C(O)—W—,        -(alkylene-O)_(n)-alkylene-J-,        -alkylene′-J-(alkylene-O)_(n)-alkylene-,        -(alkylene-O)_(n)-alkylene-J-alkylene′,        -J-(alkylene-O)_(n)-alkylene-,        -(alkylene-O)_(n)-alkylene-J-(alkylene-O)_(n)′-alkylene-J-, —W—,        -alkylene-W—, alkylene′-J-(alkylene-NMe)_(n)-alkylene-W—,        -J-(alkylene-NMe)_(n)-alkylene-W—,        -(alkylene-O)_(n)-alkylene-U-alkylene-C(O)—,        -(alkylene-O)_(n)-alkylene-U-alkylene-;        -J-alkylene-NMe-alkylene′-NMe-alkylene″-W—, and        -alkylene-J-alkylene′-NMe-alkylene″-NMe-alkylene′″-W—;        -   W has the structure of:

-   -   -   U has the structure of:

-   -   -   each J and J′ independently have the structure of:

-   -   -   each n and n′ are independently integers greater than or            equal to one; and

    -   each R₁₆ is independently selected from the group consisting of        hydrogen, halogen, alkyl, NO₂, CN, and substituted alkyl.

In some embodiments, R₁ is a polypeptide. In specific embodiments, thepolypeptide is an antibody. In certain specific embodiments, theantibody is αCD70. In other embodiments, R₂ is a polypeptide. Inspecific embodiments, the polypeptide is an antibody. In certainspecific embodiments, the antibody is αCD70.

Some embodiments of the present invention describe a compound comprisingFormula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI):

wherein:

-   -   Z has the structure of:

-   -   -   R₅ is H, CO₂H, C₁-C₆alkyl, or thiazole;        -   R₆ is OH or H;        -   Ar is phenyl or pyridine;

    -   R₁ is H, an amino protecting group, resin, at least one amino        acid, polypeptide, or polynucleotide;

    -   R₂ is OH, an ester protecting group, resin, at least one amino        acid, polypeptide, or polynucleotide;

    -   R₄ is H, halogen, lower alkyl, or substituted lower alkyl;

    -   R₇ is C₁-C₆alkyl or hydrogen;

    -   L, L₁, L₂, L₃, and L₄ are each linkers selected from the group        consisting of a bond, -alkylene-, -alkylene-C(O)—, -alkylene-J-,        -(alkylene-O)_(n)-alkylene-, -(alkylene-O)_(n)-alkylene-C(O)—,        -(alkylene-O)_(n)-J-, -(alkylene-O)_(n)-J-alkylene-,        -(alkylene-O)_(n)—(CH₂)_(n′)—NHC(O)—(CH₂)_(n″)—C(Me)₂-S—S—(CH₂)_(n′″)—NHC(O)-(alkylene-O)_(n″″)-alkylene-,        -(alkylene-O)-alkylene-W—, -alkylene-C(O)—W—,        -(alkylene-O)-alkylene-J-,        -alkylene′-J-(alkylene-O)_(n)-alkylene-,        -(alkylene-O)_(n)-alkylene-J-alkylene′,        -J-(alkylene)_(n)-alkylene-,        -(alkyleneo-O)_(n)-alkylene-J-(alkylene-O)_(n)′-alkylene-J′-,        —W—, -alkylene-W—, alkylene′-J-(alkylene-NMe)_(n)-alkylene-W—,        -J-(alkylene-NMe)_(n)-alkylene-W—,        -(alkylene-O)_(n)-alkylene-U-alkylene-C(O)—,        -(alkylene-O)_(n)-alkylene-U-alkylene-;        -J-alkylene-NMe-alkylene′-NMe-alkylene″-W—, and        -alkylene-J-alkylene′-NMe-alkylene″-NMe-alkylene′″-W—;

-   -   -   U has the structure of:

-   -   -   each J and J′ independently have the structure of:

-   -   -   each n and n′ are independently integers greater than or            equal to one; P D has the structure of:

-   -   -   each R₁₇ is independently selected from the group consisting            of H, alkyl, substituted alkyl, alkenyl, substituted            alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted            alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene            oxide, substituted polyalkylene oxide, aryl, substituted            aryl, heteroaryl, substituted heteroaryl, alkaryl,            substituted alkaryl, aralkyl, substituted aralkyl,            -(alkylene or substituted alkylene)-ON(R″)₂, -(alkylene or            substituted alkylene)-C(O)SR″, -(alkylene or substituted            alkylene)-S—S-(aryl or substituted aryl), —C(O)R″,            —C(O)₂OR″, or —C(O)N(R″)₂, wherein each R″ is independently            hydrogen, alkyl, substituted alkyl, alkenyl, substituted            alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl,            heteroaryl, alkaryl, substituted alkaryl, aralkyl, or            substituted aralkyl;        -   each Z₁ is a bond, CR₁₇R₁₇, O, S, NR′, CR₁₇R₁₇—CR₁₇R₁₇,            CR₁₇R₁₇—O, O—CR₁₇R₁₇, CR₁₇R₁₇—S, S—CR₁₇R₁₇, CR₁₇R₁₇—NR′, or            NR′—CR₁₇R₁₇;            -   each R′ is H, alkyl, or substituted alkyl;        -   each Z₂ is selected from the group consisting of a bond,            —C(O)—, —C(S)—, optionally substituted C₁-C₃ alkylene,            optionally substituted C₁-C₃ alkenylene, and optionally            substituted heteroalkyl;        -   each Z₃ are independently selected from the group consisting            of a bond, optionally substituted C₁-C₄ alkylene, optionally            substituted C₁-C₄ alkenylene, optionally substituted            heteroalkyl, —O—, —S—, —C(O)—, —C(S)—, and —N(R′)—;        -   each T₃ is a bond, C(R″)(R″), O, or S; with the proviso that            when T₃ is O or S, R″ cannot be halogen;        -   each R″ is H, halogen, alkyl, substituted alkyl, cycloalkyl,            or substituted cycloalkyl;        -   m and p are 0, 1, 2, or 3, provided that at least one of m            or p is not 0;        -   M₂ is

-   -    where (a) indicates bonding to the B group and (b) indicates        bonding to respective positions within the heterocycle group;        -   M₃ is

-   -    where (a) indicates bonding to the B group and (b) indicates        bonding to respective positions within the heterocycle group;        -   M₄ is

-   -    where (a) indicates bonding to the B group and (b) indicates        bonding to respective positions within the heterocycle group;        -   each R₁₉ is independently selected from the group consisting            of C₁-C₆ alkyl, C₁-C₆ alkoxy, ester, ether, thioether,            aminoalkyl, halogen, alkyl ester, aryl ester, amide, aryl            amide, alkyl halide, alkyl amine, alkyl sulfonic acid, alkyl            nitro, thioester, sulfonyl ester, halosulfonyl, nitrile,            alkyl nitrile, and nitro;        -   q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11; and        -   each R₁₆ is independently selected from the group consisting            of hydrogen, halogen, alkyl, NO₂, CN, and substituted alkyl.

In some embodiments, R₁ is a polypeptide. In specific embodiments, thepolypeptide is an antibody. In certain specific embodiments, theantibody is αCD70. In other embodiments, R₂ is a polypeptide. Inspecific embodiments, the polypeptide is an antibody. In certainspecific embodiments, the antibody is αCD70.

In some embodiments, a compound is described comprising Formula(XXXI-A):

In certain embodiments, a pharmaceutical composition is providedcomprising any of the compounds described and a pharmaceuticallyacceptable carrier, excipient, or binder.

In further or alternative embodiments are methods for detecting thepresence of a polypeptide in a patient, the method comprisingadministering a polypeptide comprising at least oneheterocycle-containing non-natural amino acid and the resultingheterocycle-containing non-natural amino acid polypeptide modulates theimmunogenicity of the polypeptide relative to the homologousnaturally-occurring amino acid polypeptide.

It is to be understood that the methods and compositions describedherein are not limited to the particular methodology, protocols, celllines, constructs, and reagents described herein and as such may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the methods and compositions described herein,which will be limited only by the appended claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the inventions described herein belong. Although anymethods, devices, and materials similar or equivalent to those describedherein can be used in the practice or testing of the inventionsdescribed herein, the preferred methods, devices and materials are nowdescribed.

All publications and patents mentioned herein are incorporated herein byreference in their entirety for the purpose of describing anddisclosing, for example, the constructs and methodologies that aredescribed in the publications, which might be used in connection withthe presently described inventions. The publications discussed hereinare provided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the inventors described herein are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason.

The terms “aldol-based linkage” or “mixed aldol-based linkage” refers tothe acid- or base-catalyzed condensation of one carbonyl compound withthe enolate/enol of another carbonyl compound, which may or may not bethe same, to generate a 3-hydroxy carbonyl compound—an aldol.

The term “affinity label,” as used herein, refers to a label whichreversibly or irreversibly binds another molecule, either to modify it,destroy it, or form a compound with it. By way of example, affinitylabels include enzymes and their substrates, or antibodies and theirantigens.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groups linkedto molecules via an oxygen atom, an amino group, or a sulfur atom,respectively.

The term “alkyl,” by itself or as part of another molecule means, unlessotherwise stated, a straight or branched chain, or cyclic hydrocarbonradical, or combination thereof, which may be fully saturated, mono- orpolyunsaturated and can include di- and multivalent radicals, having thenumber of carbon atoms designated (i.e. C₁-C₁₀ means one to tencarbons). Examples of saturated hydrocarbon radicals include, but arenot limited to, groups such as methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl,cyclopropylmethyl, homologs and isomers of, for example, n-pentyl,n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group isone having one or more double bonds or triple bonds. Examples ofunsaturated alkyl groups include, but are not limited to, vinyl,2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and thehigher homologs and isomers. The term “alkyl,” unless otherwise noted,is also meant to include those derivatives of alkyl defined in moredetail herein, such as “heteroalkyl”, “haloalkyl” and “homoalkyl”.

The term “alkylene” by itself or as part of another molecule means adivalent radical derived from an alkane, as exemplified, by (—CH₂—)_(n),wherein n may be 1 to about 24. By way of example only, such groupsinclude, but are not limited to, groups having 10 or fewer carbon atomssuch as the structures —CH₂CH₂— and —CH₂CH₂CH₂CH₂—. A “lower alkyl” or“lower alkylene” is a shorter chain alkyl or alkylene group, generallyhaving eight or fewer carbon atoms. The term “alkylene,” unlessotherwise noted, is also meant to include those groups described hereinas “heteroalkylene.”

The term “amino acid” refers to naturally occurring and non-naturalamino acids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally encoded amino acids are the 20 common amino acids (alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline) and pyrolysine and selenocysteine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, by way of example only, an α-carbon that is boundto a hydrogen, a carboxyl group, an amino group, and an R group. Suchanalogs may have modified R groups (by way of example, norleucine) ormay have modified peptide backbones while still retaining the same basicchemical structure as a naturally occurring amino acid. Non-limitingexamples of amino acid analogs include homoserine, norleucine,methionine sulfoxide, methionine methyl sulfonium.

Amino acids may be referred to herein by either their name, theircommonly known three letter symbols or by the one-letter symbolsrecommended by the IUPAC-IUB Biochemical Nomenclature Commission.Additionally, nucleotides, may be referred to by their commonly acceptedsingle-letter codes.

An “amino terminus modification group” refers to any molecule that canbe attached to a terminal amine group. By way of example, such terminalamine groups may be at the end of polymeric molecules, wherein suchpolymeric molecules include, but are not limited to, polypeptides,polynucleotides, and polysaccharides. Terminus modification groupsinclude but are not limited to, various water soluble polymers, peptidesor proteins. By way of example only, terminus modification groupsinclude polyethylene glycol or serum albumin. Terminus modificationgroups may be used to modify therapeutic characteristics of thepolymeric molecule, including but not limited to increasing the serumhalf-life of peptides.

By “antibody” herein is meant a protein consisting of one or morepolypeptides substantially encoded by all or part of the antibody genes.The immunoglobulin genes include, but are not limited to, the kappa,lambda, alpha, gamma (IgG1, IgG2, IgG3, and IgG4), delta, epsilon and muconstant region genes, as well as the myriad immunoglobulin variableregion genes. Antibody herein is meant to include full-length antibodiesand antibody fragments, and include antibodies that exist naturally inany organism or are engineered (e.g. are variants).

The term “antibody” refers to intact antibody, monoclonal or polyclonalantibodies. The term “antibody” also encompasses, multispecificantibodies such as bispecific antibodies. Human antibodies are usuallymade of two light chains and two heavy chains each comprising variableregions and constant regions. The light chain variable region comprises3 CDRs, identified herein as CDRL1, CDRL2 and CDRL3 flanked by frameworkregions. The heavy chain variable region comprises 3 CDRs, identifiedherein as CDRH1, CDRH2 and CDRH3 flanked by framework regions.

Anti-CD70 antibodies known in the art are suitable for use with thisinvention. Non-limiting examples of antibodies include monoclonalantibodies 2H5, 10B4, 8B5, 18E7, 69A7, 69A7Y, and 1F4 and the amino acidsequences are given in U.S. Published Patent Application 20100150950,herein incorporated by reference. The VH amino acid sequences of 2H5,10B4, 8B5, 18E7, 69A7, 69A7Y and 1F4 are shown in SEQ ID NOs:1, 2, 3, 4,5, 73, and 6 respectively. The V_(L) amino acid sequences of 2H5, 10B4,8B5, 18E7, 69A7, 69A7Y and 1F4 are shown in SEQ ID NOs:7, 8, 9, 10, 11,11, and 12, respectively of U.S. Published Patent Application20100150950 (with 69A7 and 69A7Y having the VL amino acid sequence ofSEQ ID NO: 11). Any known heavy chain sequence can be combined withlight chain sequences and in some embodiments of the present inventionthere is non-naturally encoded amino acid in the constant region of theantibodies. In some embodiments of the present invention there are twoor more non-naturally encoded amino acid in the constant region of theantibodies. Accordingly, in one aspect, this disclosure provides anisolated monoclonal antibody or antigen binding portion thereofcomprising:

(a) a heavy chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs:1, 2, 3, 4, 5, 6, and73; and

(b) a light chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, and12; and

(c) a non-naturally encoded amino acid; wherein the antibodyspecifically binds to CD70.

Anti-CD70 (αCD70) antibodies known in the art are suitable for use inthe present invention. For example, sequences for 2H5 antibody are givenin U.S. Pat. No. 8,124,738 and is incorporated herein by reference.

The term “antigen-binding fragment”, as used herein, refers to one ormore fragments of an antibody that retain the ability to bind to anantigen. It has been shown that the antigen-binding function of anantibody can be performed by fragments of an intact antibody. Examplesof binding fragments encompassed within the term “antigen-bindingfragment” of an antibody include (i) a Fab fragment, a monovalentfragment consisting of the V_(L), V_(H), C_(L) and C_(H1) domains; (ii)a F(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the V_(H) and C_(H1) domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; (vi) an isolated complementaritydetermining region (CDR), e.g., V_(H) CDRz3 comprising or not additionalsequence (linker, framework region(s) etc.) and (v) a combination of twoto six isolated CDRs comprising or not additional sequence (linker,framework region(s) etc.). Furthermore, although the two domains of theFv fragment, V_(L) and V_(H), are coded for by separate genes, they canbe joined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single polypeptide chain in which the V_(L) andV_(H) regions pair to form monovalent molecules (known as single chainFv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Hustonet al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such singlechain antibodies are also intended to be encompassed within the term“antigen-binding fragment” of an antibody. Furthermore, theantigen-binding fragments include binding-domain immunoglobulin fusionproteins comprising (i) a binding domain polypeptide (such as a heavychain variable region, a light chain variable region, or a heavy chainvariable region fused to a light chain variable region via a linkerpeptide) that is fused to an immunoglobulin hinge region polypeptide,(ii) an immunoglobulin heavy chain CH2 constant region fused to thehinge region, and (iii) an immunoglobulin heavy chain CH3 constantregion fused to the CH2 constant region. The hinge region may bemodified by replacing one or more cysteine residues with serine residuesso as to prevent dimerization. Such binding-domain immunoglobulin fusionproteins are further disclosed in US 2003/0118592 and US 2003/0133939.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are screened forutility in the same manner as are intact antibodies.

A typical antigen binding site is comprised of the variable regionsformed by the pairing of a light chain immunoglobulin and a heavy chainimmunoglobulin. The structure of the antibody variable regions is veryconsistent and exhibits very similar structures. These variable regionsare typically comprised of relatively homologous framework regions (FR)interspaced with three hypervariable regions termed ComplementarityDetermining Regions (CDRs). The overall binding activity of the antigenbinding fragment is often dictated by the sequence of the CDRs. The FRsoften play a role in the proper positioning and alignment in threedimensions of the CDRs for optimal antigen binding.

In fact, because CDR sequences are responsible for most antibody-antigeninteractions, it is possible to express recombinant antibodies thatshows the properties of specific naturally occurring antibodies byconstructing expression vectors that include CDR sequences from thespecific naturally occurring antibody grafted onto framework sequencesfrom a different antibody with different properties (see, e.g.,Riechmann, L. et al., 1998, Nature 332:323-327; Jones, P. et al., 1986,Nature 321:522-525; and Queen, C. et al., 1989, Proc. Natl. Acad. See.U.S.A. 86:10029-10033). Such framework sequences can be obtained frompublic DNA databases that include germline antibody gene sequences.These germline sequences will differ from mature antibody gene sequencesbecause they will not include completely assembled variable genes, whichare formed by V(D)J joining during B cell maturation. Germline genesequences will also differ from the sequences of a high affinitysecondary repertoire antibody which contains mutations throughout thevariable gene but typically clustered in the CDRs. For example, somaticmutations are relatively infrequent in the amino terminal portion offramework region 1 and in the carboxy-terminal portion of frameworkregion 4. Furthermore, many somatic mutations do not significantly alterthe binding properties of the antibody. For this reason, it is notnecessary to obtain the entire DNA sequence of a particular antibody inorder to recreate an intact recombinant antibody having bindingproperties similar to those of the original antibody. Partial heavy andlight chain sequence spanning the CDR regions is typically sufficientfor this purpose. The partial sequence is used to determine whichgermline variable and joining gene segments contributed to therecombined antibody variable genes. The germline sequence is then usedto fill in missing portions of the variable regions. Heavy and lightchain leader sequences are cleaved during protein maturation and do notcontribute to the properties of the final antibody. To add missingsequences, cloned cDNA sequences can be combined with syntheticoligonucleotides by ligation or PCR amplification. Alternatively, theentire variable region can be synthesized to create an entirelysynthetic variable region clone. This process has certain advantagessuch as elimination or inclusion of particular restriction sites, oroptimization of particular codons.

Of course, the totality or portions of the framework region of theantibody described herein may be used in conjunction with the CDRs inorder to optimize the affinity, specificity or any other desiredproperties of the antibody. By “antibody” herein is meant a proteinconsisting of one or more polypeptides substantially encoded by all orpart of the antibody genes. The immunoglobulin genes include, but arenot limited to, the kappa, lambda, alpha, gamma (IgG1, IgG2, IgG3, andIgG4), delta, epsilon and mu constant region genes, as well as themyriad immunoglobulin variable region genes. Antibody herein is meant toinclude full-length antibodies and antibody fragments, and includeantibodies that exist naturally in any organism or are engineered (e.g.are variants).

By “antibody fragment” is meant any form of an antibody other than thefull-length form. Antibody fragments herein include antibodies that aresmaller components that exist within full-length antibodies, andantibodies that have been engineered. Antibody fragments include but arenot limited to Fv, Fc, Fab, and (Fab′)₂, single chain Fv (scFv),diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies,CDR1, CDR2, CDR3, combinations of CDR's, variable regions, frameworkregions, constant regions, heavy chains, light chains, and variableregions, and alternative scaffold non-antibody molecules, bispecificantibodies, and the like (Maynard & Georgiou, 2000, Annu. Rev. Biomed.Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9:395-402). Anotherfunctional substructure is a single chain Fv (scFv), comprised of thevariable regions of the immunoglobulin heavy and light chain, covalentlyconnected by a peptide linker (S-z Hu et al., 1996, Cancer Research, 56,3055-3061). These small (Mr 25,000) proteins generally retainspecificity and affinity for antigen in a single polypeptide and canprovide a convenient building block for larger, antigen-specificmolecules. Unless specifically noted otherwise, statements and claimsthat use the term “antibody” or “antibodies” specifically includes“antibody fragment” and “antibody fragments.”

By “antibody-drug conjugate, or “ADC”, as used herein, refers to anantibody molecule, or fragment thereof, that is covalently bonded to oneor more biologically active molecule(s). The biologically activemolecule may be conjugated to the antibody through a linker, polymer, orother covalent bond.

The term “aromatic” or “aryl”, as used herein, refers to a closed ringstructure which has at least one ring having a conjugated pi electronsystem and includes both carbocyclic aryl and heterocyclic aryl (or“heteroaryl” or “heteroaromatic”) groups. The carbocyclic orheterocyclic aromatic group may contain from 5 to 20 ring atoms. Theterm includes monocyclic rings linked covalently or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups. An aromatic group can be unsubstituted or substituted.Non-limiting examples of “aromatic” or “aryl”, groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, anthracenyl, and phenanthracenyl.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedherein.

For brevity, the term “aromatic” or “aryl” when used in combination withother terms (including but not limited to, aryloxy, arylthioxy, aralkyl)includes both aryl and heteroaryl rings as defined above. Thus, the term“aralkyl” or “alkaryl” is meant to include those radicals in which anaryl group is attached to an alkyl group (including but not limited to,benzyl, phenethyl, pyridylmethyl and the like) including those alkylgroups in which a carbon atom (including but not limited to, a methylenegroup) has been replaced by a heteroatom, by way of example only, by anoxygen atom. Examples of such aryl groups include, but are not limitedto, phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and thelike.

The term “arylene”, as used herein, refers to a divalent aryl radical.Non-limiting examples of “arylene” include phenylene, pyridinylene,pyrimidinylene and thiophenylene. Substituents for arylene groups areselected from the group of acceptable substituents described herein.

A “bifunctional polymer”, also referred to as a “bifunctional linker”,refers to a polymer comprising two functional groups that are capable ofreacting specifically with other moieties to form covalent ornon-covalent linkages. Such moieties may include, but are not limitedto, the side groups on natural or non-natural amino acids or peptideswhich contain such natural or non-natural amino acids. The othermoieties that may be linked to the bifunctional linker or bifunctionalpolymer may be the same or different moieties. By way of example only, abifunctional linker may have a functional group reactive with a group ona first peptide, and another functional group which is reactive with agroup on a second peptide, whereby forming a conjugate that includes thefirst peptide, the bifunctional linker and the second peptide. Manyprocedures and linker molecules for attachment of various compounds topeptides are known. See, e.g., European Patent Application No. 188,256;U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338;and 4,569,789 which are incorporated by reference herein in theirentirety. A “multi-functional polymer” also referred to as a“multi-functional linker”, refers to a polymer comprising two or morefunctional groups that are capable of reacting with other moieties. Suchmoieties may include, but are not limited to, the side groups on naturalor non-natural amino acids or peptides which contain such natural ornon-natural amino acids. (including but not limited to, amino acid sidegroups) to form covalent or non-covalent linkages. A bi-functionalpolymer or multi-functional polymer may be any desired length ormolecular weight, and may be selected to provide a particular desiredspacing or conformation between one or more molecules linked to acompound and molecules it binds to or the compound.

The term “bioavailability,” as used herein, refers to the rate andextent to which a substance or its active moiety is delivered from apharmaceutical dosage form and becomes available at the site of actionor in the general circulation. Increases in bioavailability refers toincreasing the rate and extent a substance or its active moiety isdelivered from a pharmaceutical dosage form and becomes available at thesite of action or in the general circulation. By way of example, anincrease in bioavailability may be indicated as an increase inconcentration of the substance or its active moiety in the blood whencompared to other substances or active moieties. A non-limiting exampleof a method to evaluate increases in bioavailability is given inexamples 21-25. This method may be used for evaluating thebioavailability of any polypeptide.

The term “biologically active molecule”, “biologically active moiety” or“biologically active agent” when used herein means any substance whichcan affect any physical or biochemical properties of a biologicalsystem, pathway, molecule, or interaction relating to an organism,including but not limited to, viruses, bacteria, bacteriophage,transposon, prion, insects, fungi, plants, animals, and humans. Inparticular, as used herein, biologically active molecules include butare not limited to any substance intended for diagnosis, cure,mitigation, treatment, or prevention of disease in humans or otheranimals, or to otherwise enhance physical or mental well-being of humansor animals. Examples of biologically active molecules include, but arenot limited to, peptides, proteins, enzymes, small molecule drugs, harddrugs, soft drugs, prodrugs, carbohydrates, inorganic atoms ormolecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides,toxins, cells, viruses, liposomes, microparticles and micelles. Classesof biologically active agents that are suitable for use with the methodsand compositions described herein include, but are not limited to,drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics,fungicides, anti-viral agents, anti-inflammatory agents, anti-tumoragents, cardiovascular agents, anti-anxiety agents, hormones, growthfactors, steroidal agents, microbially derived toxins, and the like.

By “modulating biological activity” is meant increasing or decreasingthe reactivity of a polypeptide, altering the selectivity of thepolypeptide, enhancing or decreasing the substrate selectivity of thepolypeptide. Analysis of modified biological activity can be performedby comparing the biological activity of the non-natural polypeptide tothat of the natural polypeptide.

The term “biomaterial,” as used herein, refers to a biologically-derivedmaterial, including but not limited to material obtained frombioreactors and/or from recombinant methods and techniques.

The term “biophysical probe,” as used herein, refers to probes which candetect or monitor structural changes in molecules. Such moleculesinclude, but are not limited to, proteins and the “biophysical probe”may be used to detect or monitor interaction of proteins with othermacromolecules. Examples of biophysical probes include, but are notlimited to, spin-labels, a fluorophores, and photoactivatible groups.

The term “biosynthetically,” as used herein, refers to any methodutilizing a translation system (cellular or non-cellular), including useof at least one of the following components: a polynucleotide, a codon,a tRNA, and a ribosome. By way of example, non-natural amino acids maybe “biosynthetically incorporated” into non-natural amino acidpolypeptides using the methods and techniques described herein, “In vivogeneration of polypeptides comprising non-natural amino acids”, and inthe non-limiting example 20. Additionally, the methods for the selectionof useful non-natural amino acids which may be “biosyntheticallyincorporated” into non-natural amino acid polypeptides are described inthe non-limiting examples 20.

The term “biotin analogue,” or also referred to as “biotin mimic”, asused herein, is any molecule, other than biotin, which bind with highaffinity to avidin and/or streptavidin.

The term “carbonyl” as used herein refers to a group containing at amoiety selecting from the group consisting of —C(O)—, —S(O)—, —S(O)2-,and —C(S)—, including, but not limited to, groups containing a least oneketone group, and/or at least one aldehyde groups, and/or at least oneester group, and/or at least one carboxylic acid group, and/or at leastone thioester group. Such carbonyl groups include ketones, aldehydes,carboxylic acids, esters, and thioesters. In addition, such groups maybe part of linear, branched, or cyclic molecules.

The term “carboxy terminus modification group” refers to any moleculethat can be attached to a terminal carboxy group. By way of example,such terminal carboxy groups may be at the end of polymeric molecules,wherein such polymeric molecules include, but are not limited to,polypeptides, polynucleotides, and polysaccharides. Terminusmodification groups include but are not limited to, various watersoluble polymers, peptides or proteins. By way of example only, terminusmodification groups include polyethylene glycol or serum albumin.Terminus modification groups may be used to modify therapeuticcharacteristics of the polymeric molecule, including but not limited toincreasing the serum half-life of peptides.

The term “chemically cleavable group,” also referred to as “chemicallylabile”, as used herein, refers to a group which breaks or cleaves uponexposure to acid, base, oxidizing agents, reducing agents, chemicalinititiators, or radical initiators.

The term “chemiluminescent group,” as used herein, refers to a groupwhich emits light as a result of a chemical reaction without theaddition of heat. By way of example only, luminol(5-amino-2,3-dihydro-1,4-phthalazinedione) reacts with oxidants likehydrogen peroxide (H₂O₂) in the presence of a base and a metal catalystto produce an excited state product (3-aminophthalate, 3-APA).

The term “chromophore,” as used herein, refers to a molecule whichabsorbs light of visible wavelengths, UV wavelengths or IR wavelengths.

The term “cofactor,” as used herein, refers to an atom or moleculeessential for the action of a large molecule. Cofactors include, but arenot limited to, inorganic ions, coenzymes, proteins, or some otherfactor necessary for the activity of enzymes. Examples include, heme inhemoglobin, magnesium in chlorophyll, and metal ions for proteins.

“Cofolding,” as used herein, refers to refolding processes, reactions,or methods which employ at least two molecules which interact with eachother and result in the transformation of unfolded or improperly foldedmolecules to properly folded molecules. By way of example only,“cofolding,” employ at least two polypeptides which interact with eachother and result in the transformation of unfolded or improperly foldedpolypeptides to native, properly folded polypeptides. Such polypeptidesmay contain natural amino acids and/or at least one non-natural aminoacid.

A “comparison window,” as used herein, refers a segment of any one ofcontiguous positions used to compare a sequence to a reference sequenceof the same number of contiguous positions after the two sequences areoptimally aligned. Such contiguous positions include, but are notlimited to a group consisting of from about 20 to about 600 sequentialunits, including about 50 to about 200 sequential units, and about 100to about 150 sequential units. By way of example only, such sequencesinclude polypeptides and polypeptides containing non-natural aminoacids, with the sequential units include, but are not limited to naturaland non-natural amino acids. In addition, by way of example only, suchsequences include polynucleotides with nucleotides being thecorresponding sequential units. Methods of alignment of sequences forcomparison are well-known in the art. Optimal alignment of sequences forcomparison can be conducted, including but not limited to, by the localhomology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c,by the homology alignment algorithm of Needleman and Wunsch (1970) J.Mol. Biol. 48:443, by the search for similarity method of Pearson andLipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Ausubel et al., Current Protocols in MolecularBiology (1995 supplement)).

By way of example, an algorithm which may be used to determine percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information. TheBLAST algorithm parameters W, T, and X determine the sensitivity andspeed of the alignment. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5,N=−4 and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength of 3, and expectation (E)of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992)Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands. The BLASTalgorithm is typically performed with the “low complexity” filter turnedoff.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, or less than about0.01, or less than about 0.001.

The term “conservatively modified variants” applies to both natural andnon-natural amino acid and natural and non-natural nucleic acidsequences, and combinations thereof. With respect to particular nucleicacid sequences, “conservatively modified variants” refers to thosenatural and non-natural nucleic acids which encode identical oressentially identical natural and non-natural amino acid sequences, orwhere the natural and non-natural nucleic acid does not encode a naturaland non-natural amino acid sequence, to essentially identical sequences.By way of example, because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenprotein. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Thus by way of exampleevery natural or non-natural nucleic acid sequence herein which encodesa natural or non-natural polypeptide also describes every possiblesilent variation of the natural or non-natural nucleic acid. One ofordinary skill in the art will recognize that each codon in a natural ornon-natural nucleic acid (except AUG, which is ordinarily the only codonfor methionine, and TGG, which is ordinarily the only codon fortryptophan) can be modified to yield a functionally identical molecule.Accordingly, each silent variation of a natural and non-natural nucleicacid which encodes a natural and non-natural polypeptide is implicit ineach described sequence.

As to amino acid sequences, individual substitutions, deletions oradditions to a nucleic acid, peptide, polypeptide, or protein sequencewhich alters, adds or deletes a single natural and non-natural aminoacid or a small percentage of natural and non-natural amino acids in theencoded sequence is a “conservatively modified variant” where thealteration results in the deletion of an amino acid, addition of anamino acid, or substitution of a natural and non-natural amino acid witha chemically similar amino acid. Conservative substitution tablesproviding functionally similar natural amino acids are well known in theart. Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of themethods and compositions described herein.

Conservative substitution tables providing functionally similar aminoacids are known to those of ordinary skill in the art. The followingeight groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins:Structures and Molecular Properties (W HFreeman & Co.; 2nd edition (December 1993)

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Thus, a cycloalkylor heterocycloalkyl include saturated, partially unsaturated and fullyunsaturated ring linkages. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. The heteroatom may include, but is notlimited to, oxygen, nitrogen or sulfur. Examples of cycloalkyl include,but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkylinclude, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,2-piperazinyl, and the like. Additionally, the term encompassesmulticyclic structures, including but not limited to, bicyclic andtricyclic ring structures. Similarly, the term “heterocycloalkylene” byitself or as part of another molecule means a divalent radical derivedfrom heterocycloalkyl, and the term “cycloalkylene” by itself or as partof another molecule means a divalent radical derived from cycloalkyl.

The term “cyclodextrin,” as used herein, refers to cyclic carbohydratesconsisting of at least six to eight glucose molecules in a ringformation. The outer part of the ring contains water soluble groups; atthe center of the ring is a relatively nonpolar cavity able toaccommodate small molecules.

The term “cytotoxic,” as used herein, refers to a compound which harmscells.

“Denaturing agent” or “denaturant,” as used herein, refers to anycompound or material which will cause a reversible unfolding of apolymer. By way of example only, “denaturing agent” or “denaturants,”may cause a reversible unfolding of a protein. The strength of adenaturing agent or denaturant will be determined both by the propertiesand the concentration of the particular denaturing agent or denaturant.By way of example, denaturing agents or denaturants include, but are notlimited to, chaotropes, detergents, organic, water miscible solvents,phospholipids, or a combination thereof. Non-limiting examples ofchaotropes include, but are not limited to, urea, guanidine, and sodiumthiocyanate. Non-limiting examples of detergents may include, but arenot limited to, strong detergents such as sodium dodecyl sulfate, orpolyoxyethylene ethers (e.g. Tween or Triton detergents), Sarkosyl, mildnon-ionic detergents (e.g., digitonin), mild cationic detergents such asN→2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents(e.g. sodium cholate or sodium deoxycholate) or zwitterionic detergentsincluding, but not limited to, sulfobetaines (Zwittergent),3-(3-chlolamidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-1-propane sulfonate(CHAPSO). Non-limiting examples of organic, water miscible solventsinclude, but are not limited to, acetonitrile, lower alkanols(especially C2-C4 alkanols such as ethanol or isopropanol), or loweralkandiols (C2-C4 alkandiols such as ethylene-glycol) may be used asdenaturants. Non-limiting examples of phospholipids include, but are notlimited to, naturally occurring phospholipids such asphosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, andphosphatidylinositol or synthetic phospholipid derivatives or variantssuch as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

The term “desired functionality” as used herein refers to any groupselected from a label; a dye; a polymer; a water-soluble polymer; aderivative of polyethylene glycol; a photocrosslinker; a cytotoxiccompound; a drug; an affinity label; a photoaffinity label; a reactivecompound; a resin; a second protein or polypeptide or polypeptideanalog; an antibody or antibody fragment; a metal chelator; a cofactor;a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; anantisense polynucleotide; a saccharide, a water-soluble dendrimer, acyclodextrin, a biomaterial; a nanoparticle; a spin label; afluorophore; a metal-containing moiety; a radioactive moiety; a novelfunctional group; a group that covalently or noncovalently interactswith other molecules; a photocaged moiety; an actinic radiationexcitable moiety; a ligand; a photoisomerizable moiety; biotin; a biotinanalogue; a moiety incorporating a heavy atom; a chemically cleavablegroup; a photocleavable group; an elongated side chain; a carbon-linkedsugar; a redox-active agent; an amino thioacid; a toxic moiety; anisotopically labeled moiety; a biophysical probe; a phosphorescentgroup; a chemiluminescent group; an electron dense group; a magneticgroup; an intercalating group; a chromophore; an energy transfer agent;a biologically active agent (in which case, the biologically activeagent can include an agent with therapeutic activity and the non-naturalamino acid polypeptide or modified non-natural amino acid can serveeither as a co-therapeutic agent with the attached therapeutic agent oras a means for delivery the therapeutic agent to a desired site withinan organism); a detectable label; a small molecule; an inhibitoryribonucleic acid; a radionucleotide; a neutron-capture agent; aderivative of biotin; quantum dot(s); a nanotransmitter; aradiotransmitter; an abzyme, an activated complex activator, a virus, anadjuvant, an aglycan, an allergan, an angiostatin, an antihormone, anantioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, amacromolecule, a mimotope, a receptor, a reverse micelle, and anycombination thereof.

The term “diamine,” as used herein, refers to groups/moleculescomprising at least two amine functional groups, including, but notlimited to, a hydrazine group, an amidine group, an imine group, a1,1-diamine group, a 1,2-diamine group, a 1,3-diamine group, and a1,4-diamine group. In addition, such groups may be part of linear,branched, or cyclic molecules.

The term “detectable label,” as used herein, refers to a label which maybe observable using analytical techniques including, but not limited to,fluorescence, chemiluminescence, electron-spin resonance,ultraviolet/visible absorbance spectroscopy, mass spectrometry, nuclearmagnetic resonance, magnetic resonance, and electrochemical methods.

The term “dicarbonyl” as used herein refers to a group containing atleast two moieties selected from the group consisting of —C(O)—, —S(O)—,—S(O)₂—, and —C(S)—, including, but not limited to, 1,2-dicarbonylgroups, a 1,3-dicarbonyl groups, and 1,4-dicarbonyl groups, and groupscontaining a least one ketone group, and/or at least one aldehydegroups, and/or at least one ester group, and/or at least one carboxylicacid group, and/or at least one thioester group. Such dicarbonyl groupsinclude diketones, ketoaldehydes, ketoacids, ketoesters, andketothioesters. In addition, such groups may be part of linear,branched, or cyclic molecules. The two moieties in the dicarbonyl groupmay be the same or different, and may include substituents that wouldproduce, by way of example only, an ester, a ketone, an aldehyde, athioester, or an amide, at either of the two moieties.

The term “drug,” as used herein, refers to any substance used in theprevention, diagnosis, alleviation, treatment, or cure of a disease orcondition.

The term “dye,” as used herein, refers to a soluble, coloring substancewhich contains a chromophore.

The term “effective amount,” as used herein, refers to a sufficientamount of an agent or a compound being administered which will relieveto some extent one or more of the symptoms of the disease or conditionbeing treated. The result can be reduction and/or alleviation of thesigns, symptoms, or causes of a disease, or any other desired alterationof a biological system. By way of example, an agent or a compound beingadministered includes, but is not limited to, a natural amino acidpolypeptide, non-natural amino acid polypeptide, modified natural aminoacid polypeptide, or modified non-amino acid polypeptide. Compositionscontaining such natural amino acid polypeptides, non-natural amino acidpolypeptides, modified natural amino acid polypeptides, or modifiednon-natural amino acid polypeptides can be administered forprophylactic, enhancing, and/or therapeutic treatments. An appropriate“effective” amount in any individual case may be determined usingtechniques, such as a dose escalation study.

The term “electron dense group,” as used herein, refers to a group whichscatters electrons when irradiated with an electron beam. Such groupsinclude, but are not limited to, ammonium molybdate, bismuth subnitratecadmium iodide, 99%, carbohydrazide, ferric chloride hexahydrate,hexamethylene tetramine, 98.5%, indium trichloride anhydrous, lanthanumnitrate, lead acetate trihydrate, lead citrate trihydrate, lead nitrate,periodic acid, phosphomolybdic acid, phosphotungstic acid, potassiumferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate,silver proteinate (Ag Assay: 8.0-8.5%) “Strong”, silvertetraphenylporphin (S-TPPS), sodium chloroaurate, sodium tungstate,thallium nitrate, thiosemicarbazide (TSC), uranyl acetate, uranylnitrate, and vanadyl sulfate.

The term “energy transfer agent,” as used herein, refers to a moleculewhich can either donate or accept energy from another molecule. By wayof example only, fluorescence resonance energy transfer (FRET) is adipole-dipole coupling process by which the excited-state energy of afluorescence donor molecule is non-radiatively transferred to anunexcited acceptor molecule which then fluorescently emits the donatedenergy at a longer wavelength.

The terms “enhance” or “enhancing” means to increase or prolong eitherin potency or duration a desired effect. By way of example, “enhancing”the effect of therapeutic agents refers to the ability to increase orprolong, either in potency or duration, the effect of therapeutic agentson during treatment of a disease, disorder or condition. An“enhancing-effective amount,” as used herein, refers to an amountadequate to enhance the effect of a therapeutic agent in the treatmentof a disease, disorder or condition. When used in a patient, amountseffective for this use will depend on the severity and course of thedisease, disorder or condition, previous therapy, the patient's healthstatus and response to the drugs, and the judgment of the treatingphysician.

As used herein, the term “eukaryote” refers to organisms belonging tothe phylogenetic domain Eucarya, including but not limited to animals(including but not limited to, mammals, insects, reptiles, birds, etc.),ciliates, plants (including but not limited to, monocots, dicots, andalgae), fungi, yeasts, flagellates, microsporidia, and protists.

The term “fatty acid,” as used herein, refers to carboxylic acids withabout C6 or longer hydrocarbon side chain.

The term “fluorophore,” as used herein, refers to a molecule which uponexcitation emits photons and is thereby fluorescent.

The terms “functional group”, “active moiety”, “activating group”,“leaving group”, “reactive site”, “chemically reactive group” and“chemically reactive moiety,” as used herein, refer to portions or unitsof a molecule at which chemical reactions occur. The terms are somewhatsynonymous in the chemical arts and are used herein to indicate theportions of molecules that perform some function or activity and arereactive with other molecules.

The term “halogen” includes fluorine, chlorine, iodine, and bromine.

The term “haloacyl,” as used herein, refers to acyl groups which containhalogen moieties, including, but not limited to, —C(O)CH₃, —C(O)CF₃,—C(O)CH₂OCH₃, and the like.

The term “haloalkyl,” as used herein, refers to alkyl groups whichcontain halogen moieties, including, but not limited to, —CF₃ and—CH₂CF₃ and the like.

The term “heteroalkyl,” as used herein, refers to straight or branchedchain, or cyclic hydrocarbon radicals, or combinations thereof,consisting of an alkyl group and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. In addition, up to two heteroatoms may beconsecutive, such as, by way of example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃.

The terms “heterocyclic-based linkage” or “heterocycle linkage” refersto a moiety formed from the reaction of a dicarbonyl group with adiamine group. The resulting reaction product is a heterocycle,including a heteroaryl group or a heterocycloalkyl group. The resultingheterocycle group serves as a chemical link between a non-natural aminoacid or non-natural amino acid polypeptide and another functional group.In one embodiment, the heterocycle linkage includes anitrogen-containing heterocycle linkage, including by way of exampleonly a pyrazole linkage, a pyrrole linkage, an indole linkage, abenzodiazepine linkage, and a pyrazalone linkage.

Similarly, the term “heteroalkylene” refers to a divalent radicalderived from heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, the same or different heteroatoms can also occupy either or bothof the chain termini (including but not limited to, alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, and thelike). Still further, for alkylene and heteroalkylene linking groups, noorientation of the linking group is implied by the direction in whichthe formula of the linking group is written. By way of example, theformula —C(O)₂R′— represents both —C(O)₂R′— and —R′C(O)₂—.

The term “heteroaryl” or “heteroaromatic,” as used herein, refers toaryl groups which contain at least one heteroatom selected from N, O,and S; wherein the nitrogen and sulfur atoms may be optionally oxidized,and the nitrogen atom(s) may be optionally quaternized. Heteroarylgroups may be substituted or unsubstituted. A heteroaryl group may beattached to the remainder of the molecule through a heteroatom.Non-limiting examples of heteroaryl groups include 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl,pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.

The term “homoalkyl,” as used herein refers to alkyl groups which arehydrocarbon groups.

The term “identical,” as used herein, refers to two or more sequences orsubsequences which are the same. In addition, the term “substantiallyidentical,” as used herein, refers to two or more sequences which have apercentage of sequential units which are the same when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using comparison algorithms or by manualalignment and visual inspection. By way of example only, two or moresequences may be “substantially identical” if the sequential units areabout 60% identical, about 65% identical, about 70% identical, about 75%identical, about 80% identical, about 85% identical, about 90%identical, or about 95% identical over a specified region. Suchpercentages to describe the “percent identity” of two or more sequences.The identity of a sequence can exist over a region that is at leastabout 75-100 sequential units in length, over a region that is about 50sequential units in length, or, where not specified, across the entiresequence. This definition also refers to the complement of a testsequence. By way of example only, two or more polypeptide sequences areidentical when the amino acid residues are the same, while two or morepolypeptide sequences are “substantially identical” if the amino acidresidues are about 60% identical, about 65% identical, about 70%identical, about 75% identical, about 80% identical, about 85%identical, about 90% identical, or about 95% identical over a specifiedregion. The identity can exist over a region that is at least about 75to about 100 amino acids in length, over a region that is about 50 aminoacids in length, or, where not specified, across the entire sequence ofa polypeptide sequence. In addition, by way of example only, two or morepolynucleotide sequences are identical when the nucleic acid residuesare the same, while two or more polynucleotide sequences are“substantially identical” if the nucleic acid residues are about 60%identical, about 65% identical, about 70% identical, about 75%identical, about 80% identical, about 85% identical, about 90%identical, or about 95% identical over a specified region. The identitycan exist over a region that is at least about 75 to about 100 nucleicacids in length, over a region that is about 50 nucleic acids in length,or, where not specified, across the entire sequence of a polynucleotidesequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

The term “immunogenicity,” as used herein, refers to an antibodyresponse to administration of a therapeutic drug. The immunogenicitytoward therapeutic non-natural amino acid polypeptides can be obtainedusing quantitative and qualitative assays for detection ofanti-non-natural amino acid polypeptides antibodies in biologicalfluids. Such assays include, but are not limited to, Radioimmunoassay(RIA), Enzyme-linked immunosorbent assay (ELISA), luminescentimmunoassay (LIA), and fluorescent immunoassay (FIA). Analysis ofimmunogenicity toward therapeutic non-natural amino acid polypeptidesinvolves comparing the antibody response upon administration oftherapeutic non-natural amino acid polypeptides to the antibody responseupon administration of therapeutic natural amino acid polypeptides.

The term “intercalating agent,” also referred to as “intercalatinggroup,” as used herein, refers to a chemical that can insert into theintramolecular space of a molecule or the intermolecular space betweenmolecules. By way of example only an intercalating agent or group may bea molecule which inserts into the stacked bases of the DNA double helix.

The term “isolated,” as used herein, refers to separating and removing acomponent of interest from components not of interest. Isolatedsubstances can be in either a dry or semi-dry state, or in solution,including but not limited to an aqueous solution. The isolated componentcan be in a homogeneous state or the isolated component can be a part ofa pharmaceutical composition that comprises additional pharmaceuticallyacceptable carriers and/or excipients. Purity and homogeneity may bedetermined using analytical chemistry techniques including, but notlimited to, polyacrylamide gel electrophoresis or high performanceliquid chromatography. In addition, when a component of interest isisolated and is the predominant species present in a preparation, thecomponent is described herein as substantially purified. The term“purified,” as used herein, may refer to a component of interest whichis at least 85% pure, at least 90% pure, at least 95% pure, at least 99%or greater pure. By way of example only, nucleic acids or proteins are“isolated” when such nucleic acids or proteins are free of at least someof the cellular components with which it is associated in the naturalstate, or that the nucleic acid or protein has been concentrated to alevel greater than the concentration of its in vivo or in vitroproduction. Also, by way of example, a gene is isolated when separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest.

The term “label,” as used herein, refers to a substance which isincorporated into a compound and is readily detected, whereby itsphysical distribution may be detected and/or monitored.

The term “linkage,” as used herein to refer to bonds or chemical moietyformed from a chemical reaction between the functional group of a linkerand another molecule. Such bonds may include, but are not limited to,covalent linkages and non-covalent bonds, while such chemical moietiesmay include, but are not limited to, esters, carbonates, iminesphosphate esters, hydrazones, acetals, orthoesters, peptide linkages,and oligonucleotide linkages. Hydrolytically stable linkages means thatthe linkages are substantially stable in water and do not react withwater at useful pH values, including but not limited to, underphysiological conditions for an extended period of time, perhaps evenindefinitely. Hydrolytically unstable or degradable linkages means thatthe linkages are degradable in water or in aqueous solutions, includingfor example, blood. Enzymatically unstable or degradable linkages meansthat the linkage can be degraded by one or more enzymes. By way ofexample only, PEG and related polymers may include degradable linkagesin the polymer backbone or in the linker group between the polymerbackbone and one or more of the terminal functional groups of thepolymer molecule. Such degradable linkages include, but are not limitedto, ester linkages formed by the reaction of PEG carboxylic acids oractivated PEG carboxylic acids with alcohol groups on a biologicallyactive agent, wherein such ester groups generally hydrolyze underphysiological conditions to release the biologically active agent. Otherhydrolytically degradable linkages include but are not limited tocarbonate linkages; imine linkages resulted from reaction of an amineand an aldehyde; phosphate ester linkages formed by reacting an alcoholwith a phosphate group; hydrazone linkages which are reaction product ofa hydrazide and an aldehyde; acetal linkages that are the reactionproduct of an aldehyde and an alcohol; orthoester linkages that are thereaction product of a formate and an alcohol; peptide linkages formed byan amine group, including but not limited to, at an end of a polymersuch as PEG, and a carboxyl group of a peptide; and oligonucleotidelinkages formed by a phosphoramidite group, including but not limitedto, at the end of a polymer, and a 5′ hydroxyl group of anoligonucleotide.

The terms “medium” or “media,” as used herein, refer to any culturemedium used to grow and harvest cells and/or products expressed and/orsecreted by such cells. Such “medium” or “media” include, but are notlimited to, solution, solid, semi-solid, or rigid supports that maysupport or contain any host cell, including, by way of example,bacterial host cells, yeast host cells, insect host cells, plant hostcells, eukaryotic host cells, mammalian host cells, CHO cells,prokaryotic host cells, E. coli, or Pseudomonas host cells, and cellcontents. Such “medium” or “media” includes, but is not limited to,medium or media in which the host cell has been grown into which apolypeptide has been secreted, including medium either before or after aproliferation step. Such “medium” or “media” also includes, but is notlimited to, buffers or reagents that contain host cell lysates, by wayof example a polypeptide produced intracellularly and the host cells arelysed or disrupted to release the polypeptide.

The term “metabolite,” as used herein, refers to a derivative of acompound, by way of example natural amino acid polypeptide, anon-natural amino acid polypeptide, a modified natural amino acidpolypeptide, or a modified non-natural amino acid polypeptide, that isformed when the compound, by way of example natural amino acidpolypeptide, non-natural amino acid polypeptide, modified natural aminoacid polypeptide, or modified non-natural amino acid polypeptide, ismetabolized. The term “pharmaceutically active metabolite” or “activemetabolite” refers to a biologically active derivative of a compound, byway of example natural amino acid polypeptide, a non-natural amino acidpolypeptide, a modified natural amino acid polypeptide, or a modifiednon-natural amino acid polypeptide, that is formed when such a compound,by way of example a natural amino acid polypeptide, non-natural aminoacid polypeptide, modified natural amino acid polypeptide, or modifiednon-natural amino acid polypeptide, is metabolized.

The term “metabolized,” as used herein, refers to the sum of theprocesses by which a particular substance is changed by an organism.Such processes include, but are not limited to, hydrolysis reactions andreactions catalyzed by enzymes. Further information on metabolism may beobtained from The Pharmacological Basis of Therapeutics, 9th Edition,McGraw-Hill (1996). By way of example only, metabolites of natural aminoacid polypeptides, non-natural amino acid polypeptides, modified naturalamino acid polypeptides, or modified non-natural amino acid polypeptidesmay be identified either by administration of the natural amino acidpolypeptides, non-natural amino acid polypeptides, modified naturalamino acid polypeptides, or modified non-natural amino acid polypeptidesto a host and analysis of tissue samples from the host, or by incubationof natural amino acid polypeptides, non-natural amino acid polypeptides,modified natural amino acid polypeptides, or modified non-natural aminoacid polypeptides with hepatic cells in vitro and analysis of theresulting compounds.

The term “metal chelator,” as used herein, refers to a molecule whichforms a metal complex with metal ions. By way of example, such moleculesmay form two or more coordination bonds with a central metal ion and mayform ring structures.

The term “metal-containing moiety,” as used herein, refers to a groupwhich contains a metal ion, atom or particle. Such moieties include, butare not limited to, cisplatin, chelated metals ions (such as nickel,iron, and platinum), and metal nanoparticles (such as nickel, iron, andplatinum).

The term “moiety incorporating a heavy atom,” as used herein, refers toa group which incorporates an ion of atom which is usually heavier thancarbon. Such ions or atoms include, but are not limited to, silicon,tungsten, gold, lead, and uranium.

The term “modified,” as used herein refers to the presence of a changeto a natural amino acid, a non-natural amino acid, a natural amino acidpolypeptide or a non-natural amino acid polypeptide. Such changes, ormodifications, may be obtained by post synthesis modifications ofnatural amino acids, non-natural amino acids, natural amino acidpolypeptides or non-natural amino acid polypeptides, or byco-translational, or by post-translational modification of natural aminoacids, non-natural amino acids, natural amino acid polypeptides ornon-natural amino acid polypeptides. The form “modified or unmodified”means that the natural amino acid, non-natural amino acid, natural aminoacid polypeptide or non-natural amino acid polypeptide being discussedare optionally modified, that is, the natural amino acid, non-naturalamino acid, natural amino acid polypeptide or non-natural amino acidpolypeptide under discussion can be modified or unmodified.

As used herein, the term “modulated serum half-life” refers to positiveor negative changes in the circulating half-life of a modifiedbiologically active molecule relative to its non-modified form. By wayof example, the modified biologically active molecules include, but arenot limited to, natural amino acid, non-natural amino acid, naturalamino acid polypeptide or non-natural amino acid polypeptide. By way ofexample, serum half-life is measured by taking blood samples at varioustime points after administration of the biologically active molecule ormodified biologically active molecule, and determining the concentrationof that molecule in each sample. Correlation of the serum concentrationwith time allows calculation of the serum half-life. By way of example,modulated serum half-life may be an increased in serum half-life, whichmay enable an improved dosing regimens or avoid toxic effects. Suchincreases in serum may be at least about two fold, at least aboutthree-fold, at least about five-fold, or at least about ten-fold.Methods for evaluating serum half-life are known in the art and may beused for evaluating the serum half-life of antibodies and antibody drugconjugates of the present invention.

The term “modulated therapeutic half-life,” as used herein, refers topositive or negative change in the half-life of the therapeuticallyeffective amount of a modified biologically active molecule, relative toits non-modified form. By way of example, the modified biologicallyactive molecules include, but are not limited to, natural amino acid,non-natural amino acid, natural amino acid polypeptide or non-naturalamino acid polypeptide. By way of example, therapeutic half-life ismeasured by measuring pharmacokinetic and/or pharmacodynamic propertiesof the molecule at various time points after administration. Increasedtherapeutic half-life may enable a particular beneficial dosing regimen,a particular beneficial total dose, or avoids an undesired effect. Byway of example, the increased therapeutic half-life may result fromincreased potency, increased or decreased binding of the modifiedmolecule to its target, an increase or decrease in another parameter ormechanism of action of the non-modified molecule, or an increased ordecreased breakdown of the molecules by enzymes such as, by way ofexample only, proteases. Methods for evaluating therapeutic half-lifeare known in the art and may be used for evaluating the therapeutichalf-life of antibodies and antibody drug conjugates of the presentinvention.

The term “nanoparticle,” as used herein, refers to a particle which hasa particle size between about 500 nm to about 1 nm.

The term “near-stoichiometric,” as used herein, refers to the ratio ofthe moles of compounds participating in a chemical reaction being about0.75 to about 1.5.

As used herein, the term “non-eukaryote” refers to non-eukaryoticorganisms. By way of example, a non-eukaryotic organism may belong tothe Eubacteria, (which includes but is not limited to, Escherichia coli,Thermus thermophilus, or Bacillus stearothermophilus, Pseudomonasfluorescens, Pseudomonas aeruginosa, Pseudomonas putida), phylogeneticdomain, or the Archaea, which includes, but is not limited to,Methanococcus jannaschii, Methanobacterium thermoautotrophicum,Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii,Aeuropyrum pernix, or Halobacterium such as Haloferax volcanii andHalobacterium species NRC-1, or phylogenetic domain.

A “non-natural amino acid” refers to an amino acid that is not one ofthe 20 common amino acids or pyrolysine or selenocysteine. Other termsthat may be used synonymously with the term “non-natural amino acid” is“non-naturally encoded amino acid,” “unnatural amino acid,”“non-naturally-occurring amino acid,” and variously hyphenated andnon-hyphenated versions thereof. The term “non-natural amino acid”includes, but is not limited to, amino acids which occur naturally bymodification of a naturally encoded amino acid (including but notlimited to, the 20 common amino acids or pyrrolysine and selenocysteine)but are not themselves incorporated into a growing polypeptide chain bythe translation complex. Examples of naturally-occurring amino acidsthat are not naturally-encoded include, but are not limited to,N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, andO-phosphotyrosine. Additionally, the term “non-natural amino acid”includes, but is not limited to, amino acids which do not occurnaturally and may be obtained synthetically or may be obtained bymodification of non-natural amino acids.

The term “nucleic acid,” as used herein, refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides or ribonucleotides and polymersthereof in either single- or double-stranded form. By way of exampleonly, such nucleic acids and nucleic acid polymers include, but are notlimited to, (i) analogues of natural nucleotides which have similarbinding properties as a reference nucleic acid and are metabolized in amanner similar to naturally occurring nucleotides; (ii) oligonucleotideanalogs including, but are not limited to, PNA (peptidonucleic acid),analogs of DNA used in antisense technology (phosphorothioates,phosphoroamidates, and the like); (iii) conservatively modified variantsthereof (including but not limited to, degenerate codon substitutions)and complementary sequences and sequence explicitly indicated. By way ofexample, degenerate codon substitutions may be achieved by generatingsequences in which the third position of one or more selected (or all)codons is substituted with mixed-base and/or deoxyinosine residues(Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J.Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell.Probes 8:91-98 (1994)).

The term “oxidizing agent,” as used herein, refers to a compound ormaterial which is capable of removing an electron from a compound beingoxidized. By way of example oxidizing agents include, but are notlimited to, oxidized glutathione, cystine, cystamine, oxidizeddithiothreitol, oxidized erythreitol, and oxygen. A wide variety ofoxidizing agents are suitable for use in the methods and compositionsdescribed herein.

The term “pharmaceutically acceptable”, as used herein, refers to amaterial, including but not limited, to a salt, carrier or diluent,which does not abrogate the biological activity or properties of thecompound, and is relatively nontoxic, i.e., the material may beadministered to an individual without causing undesirable biologicaleffects or interacting in a deleterious manner with any of thecomponents of the composition in which it is contained.

The term “photoaffinity label,” as used herein, refers to a label with agroup, which, upon exposure to light, forms a linkage with a moleculefor which the label has an affinity. By way of example only, such alinkage may be covalent or non-covalent.

The term “photocaged moiety,” as used herein, refers to a group which,upon illumination at certain wavelengths, covalently or non-covalentlybinds other ions or molecules.

The term “photocleavable group,” as used herein, refers to a group whichbreaks upon exposure to light.

The term “photocrosslinker,” as used herein, refers to a compoundcomprising two or more functional groups which, upon exposure to light,are reactive and form a covalent or non-covalent linkage with two ormore monomeric or polymeric molecules.

The term “photoisomerizable moiety,” as used herein, refers to a groupwherein upon illumination with light changes from one isomeric form toanother.

The term “polyalkylene glycol,” as used herein, refers to linear orbranched polymeric polyether polyols. Such polyalkylene glycols,including, but are not limited to, polyethylene glycol, polypropyleneglycol, polybutylene glycol, and derivatives thereof. Other exemplaryembodiments are listed, for example, in commercial supplier catalogs,such as Shearwater Corporation's catalog “Polyethylene Glycol andDerivatives for Biomedical Applications” (2001). By way of example only,such polymeric polyether polyols have average molecular weights betweenabout 0.1 kDa to about 100 kDa. By way of example, such polymericpolyether polyols include, but are not limited to, between about 100 Daand about 100,000 Da or more. The molecular weight of the polymer may bebetween about 100 Da and about 100,000 Da, including but not limited to,about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da,about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da,about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da,about 40,000 Da, about 35,000 Da, about 30,000 Da, about 25,000 Da,about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000Da, about 3,000 Da, about 2,000 Da, about 1,000 Da, about 900 Da, about800 Da, about 700 Da, about 600 Da, about 500 Da, 400 Da, about 300 Da,about 200 Da, and about 100 Da. In some embodiments molecular weight ofthe polymer is between about 100 Da and about 50,000 Da. In someembodiments, the molecular weight of the polymer is between about 100 Daand about 40,000 Da. In some embodiments, the molecular weight of thepolymer is between about 1,000 Da and about 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 2,000to about 50,000 Da. In some embodiments, the molecular weight of thepolymer is between about 5,000 Da and about 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 10,000Da and about 40,000 Da. In some embodiments, the poly(ethylene glycol)molecule is a branched polymer. The molecular weight of the branchedchain PEG may be between about 1,000 Da and about 100,000 Da, includingbut not limited to, about 100,000 Da, about 95,000 Da, about 90,000 Da,about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da,about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da,about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da,about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da,about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, and about1,000 Da. In some embodiments, the molecular weight of the branchedchain PEG is between about 1,000 Da and about 50,000 Da. In someembodiments, the molecular weight of the branched chain PEG is betweenabout 1,000 Da and about 40,000 Da. In some embodiments, the molecularweight of the branched chain PEG is between about 5,000 Da and about40,000 Da. In some embodiments, the molecular weight of the branchedchain PEG is between about 5,000 Da and about 20,000 Da. In otherembodiments, the molecular weight of the branched chain PEG is betweenabout 2,000 to about 50,000 Da.

The term “polymer,” as used herein, refers to a molecule composed ofrepeated subunits. Such molecules include, but are not limited to,polypeptides, polynucleotides, or polysaccharides or polyalkyleneglycols.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-natural amino acid. Additionally, such “polypeptides,” “peptides”and “proteins” include amino acid chains of any length, including fulllength proteins, wherein the amino acid residues are linked by covalentpeptide bonds.

The term “post-translationally modified” refers to any modification of anatural or non-natural amino acid which occurs after such an amino acidhas been translationally incorporated into a polypeptide chain. Suchmodifications include, but are not limited to, co-translational in vivomodifications, co-translational in vitro modifications (such as in acell-free translation system), post-translational in vivo modifications,and post-translational in vitro modifications.

The terms “prodrug” or “pharmaceutically acceptable prodrug,” as usedherein, refers to an agent that is converted into the parent drug invivo or in vitro, wherein which does not abrogate the biologicalactivity or properties of the drug, and is relatively nontoxic, i.e.,the material may be administered to an individual without causingundesirable biological effects or interacting in a deleterious mannerwith any of the components of the composition in which it is contained.Prodrugs are generally drug precursors that, following administration toa subject and subsequent absorption, are converted to an active, or amore active species via some process, such as conversion by a metabolicpathway. Some prodrugs have a chemical group present on the prodrug thatrenders it less active and/or confers solubility or some other propertyto the drug. Once the chemical group has been cleaved and/or modifiedfrom the prodrug the active drug is generated. Prodrugs are convertedinto active drug within the body through enzymatic or non-enzymaticreactions. Prodrugs may provide improved physiochemical properties suchas better solubility, enhanced delivery characteristics, such asspecifically targeting a particular cell, tissue, organ or ligand, andimproved therapeutic value of the drug. The benefits of such prodrugsinclude, but are not limited to, (i) ease of administration comparedwith the parent drug; (ii) the prodrug may be bioavailable by oraladministration whereas the parent is not; and (iii) the prodrug may alsohave improved solubility in pharmaceutical compositions compared withthe parent drug. A pro-drug includes a pharmacologically inactive, orreduced-activity, derivative of an active drug. Prodrugs may be designedto modulate the amount of a drug or biologically active molecule thatreaches a desired site of action through the manipulation of theproperties of a drug, such as physiochemical, biopharmaceutical, orpharmacokinetic properties. An example, without limitation, of a prodrugwould be a non-natural amino acid polypeptide which is administered asan ester (the “prodrug”) to facilitate transmittal across a cellmembrane where water solubility is detrimental to mobility but whichthen is metabolically hydrolyzed to the carboxylic acid, the activeentity, once inside the cell where water solubility is beneficial.Prodrugs may be designed as reversible drug derivatives, for use asmodifiers to enhance drug transport to site-specific tissues.

The term “prophylactically effective amount,” as used herein, refersthat amount of a composition containing at least one non-natural aminoacid polypeptide or at least one modified non-natural amino acidpolypeptide prophylactically applied to a patient which will relieve tosome extent one or more of the symptoms of a disease, condition ordisorder being treated. In such prophylactic applications, such amountsmay depend on the patient's state of health, weight, and the like. It isconsidered well within the skill of the art for one to determine suchprophylactically effective amounts by routine experimentation,including, but not limited to, a dose escalation clinical trial.

The term “protected,” as used herein, refers to the presence of a“protecting group” or moiety that prevents reaction of the chemicallyreactive functional group under certain reaction conditions. Theprotecting group will vary depending on the type of chemically reactivegroup being protected. By way of example only, (i) if the chemicallyreactive group is an amine or a hydrazide, the protecting group may beselected from tert-butyloxycarbonyl (t-Boc) and9-fluorenylmethoxycarbonyl (Fmoc); (ii) if the chemically reactive groupis a thiol, the protecting group may be orthopyridyldisulfide; and (iii)if the chemically reactive group is a carboxylic acid, such as butanoicor propionic acid, or a hydroxyl group, the protecting group may bebenzyl or an alkyl group such as methyl, ethyl, or tert-butyl.

By way of example only, blocking/protecting groups may be selected from:

Additionally, protecting groups include, but are not limited to,including photolabile groups such as Nvoc and MeNvoc and otherprotecting groups known in the art. Other protecting groups aredescribed in Greene and Wuts, Protective Groups in Organic Synthesis,3rd Ed., John Wiley & Sons, New York, N.Y., 1999, which is incorporatedherein by reference in its entirety.

The term “radioactive moiety,” as used herein, refers to a group whosenuclei spontaneously give off nuclear radiation, such as alpha, beta, orgamma particles; wherein, alpha particles are helium nuclei, betaparticles are electrons, and gamma particles are high energy photons.

The term “reactive compound,” as used herein, refers to a compound whichunder appropriate conditions is reactive toward another atom, moleculeor compound.

The term “recombinant host cell,” also referred to as “host cell,”refers to a cell which includes an exogenous polynucleotide, wherein themethods used to insert the exogenous polynucleotide into a cell include,but are not limited to, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. By way ofexample only, such exogenous polynucleotide may be a nonintegratedvector, including but not limited to a plasmid, or may be integratedinto the host genome.

The term “redox-active agent,” as used herein, refers to a moleculewhich oxidizes or reduces another molecule, whereby the redox activeagent becomes reduced or oxidized. Examples of redox active agentinclude, but are not limited to, ferrocene, quinones, Ru^(2+/3+)complexes, Co^(2+/+) complexes, and Os^(2+/3+) complexes.

The term “reducing agent,” as used herein, refers to a compound ormaterial which is capable of adding an electron to a compound beingreduced. By way of example reducing agents include, but are not limitedto, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine,cysteoamine (2-aminoethanethiol), and reduced glutathione. Such reducingagents may be used, by way of example only, to maintain sulfhydrylgroups in the reduced state and to reduce intra- or intermoleculardisulfide bonds.

“Refolding,” as used herein describes any process, reaction or methodwhich transforms an improperly folded or unfolded state to a native orproperly folded conformation. By way of example only, refoldingtransforms disulfide bond containing polypeptides from an improperlyfolded or unfolded state to a native or properly folded conformationwith respect to disulfide bonds. Such disulfide bond containingpolypeptides to may be natural amino acid polypeptides or non-naturalamino acid polypeptides.

The term “resin,” as used herein, refers to high molecular weight,insoluble polymer beads. By way of example only, such beads may be usedas supports for solid phase peptide synthesis, or sites for attachmentof molecules prior to purification.

The term “saccharide,” as used herein, refers to a series ofcarbohydrates including but not limited to sugars, monosaccharides,oligosaccharides, and polysaccharides.

The term “safety” or “safety profile,” as used herein, refers to sideeffects that might be related to administration of a drug relative tothe number of times the drug has been administered. By way of example, adrug which has been administered many times and produced only mild or noside effects is said to have an excellent safety profile. A non-limitingexample of a method to evaluate the safety profile is given in example26. This method may be used for evaluating the safety profile of anypolypeptide.

The phrase “selectively hybridizes to” or “specifically hybridizes to,”as used herein, refers to the binding, duplexing, or hybridizing of amolecule to a particular nucleotide sequence under stringenthybridization conditions when that sequence is present in a complexmixture including but not limited to, total cellular or library DNA orRNA.

The term “spin label,” as used herein, refers to molecules which containan atom or a group of atoms exhibiting an unpaired electron spin (i.e. astable paramagnetic group) that can be detected by electron spinresonance spectroscopy and can be attached to another molecule. Suchspin-label molecules include, but are not limited to, nitryl radicalsand nitroxides, and may be single spin-labels or double spin-labels.

The term “stoichiometric,” as used herein, refers to the ratio of themoles of compounds participating in a chemical reaction being about 0.9to about 1.1.

The term “stoichiometric-like,” as used herein, refers to a chemicalreaction which becomes stoichiometric or near-stoichiometric uponchanges in reaction conditions or in the presence of additives. Suchchanges in reaction conditions include, but are not limited to, anincrease in temperature or change in pH. Such additives include, but arenot limited to, accelerants.

The phrase “stringent hybridization conditions” refers to hybridizationof sequences of DNA, RNA, PNA or other nucleic acid mimics, orcombinations thereof, under conditions of low ionic strength and hightemperature. By way of example, under stringent conditions a probe willhybridize to its target subsequence in a complex mixture of nucleic acid(including but not limited to, total cellular or library DNA or RNA) butdoes not hybridize to other sequences in the complex mixture. Stringentconditions are sequence-dependent and will be different in differentcircumstances. By way of example, longer sequences hybridizespecifically at higher temperatures. Stringent hybridization conditionsinclude, but are not limited to, (i) about 5-10° C. lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH; (ii) the salt concentration is about 0.01 M to about1.0 M at about pH 7.0 to about pH 8.3 and the temperature is at leastabout 30° C. for short probes (including but not limited to, about 10 toabout 50 nucleotides) and at least about 60° C. for long probes(including but not limited to, greater than 50 nucleotides); (iii) theaddition of destabilizing agents including, but not limited to,formamide, (iv) 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C.,or 5×SSC, about 1% SDS, incubating at 65° C., with wash in 0.2×SSC, andabout 0.1% SDS at 65° C. for between about 5 minutes to about 120minutes. By way of example only, detection of selective or specifichybridization, includes, but is not limited to, a positive signal atleast two times background. An extensive guide to the hybridization ofnucleic acids is found in Tijssen, Laboratory Techniques in Biochemistryand Molecular Biology—Hybridization with Nucleic Probes, “Overview ofprinciples of hybridization and the strategy of nucleic acid assays”(1993).

The term “subject” as used herein, refers to an animal which is theobject of treatment, observation or experiment. By way of example only,a subject may be, but is not limited to, a mammal including, but notlimited to, a human.

The term “substantially purified,” as used herein, refers to a componentof interest that may be substantially or essentially free of othercomponents which normally accompany or interact with the component ofinterest prior to purification. By way of example only, a component ofinterest may be “substantially purified” when the preparation of thecomponent of interest contains less than about 30%, less than about 25%,less than about 20%, less than about 15%, less than about 10%, less thanabout 5%, less than about 4%, less than about 3%, less than about 2%, orless than about 1% (by dry weight) of contaminating components. Thus, a“substantially purified” component of interest may have a purity levelof about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,about 96%, about 97%, about 98%, about 99% or greater. By way of exampleonly, a natural amino acid polypeptide or a non-natural amino acidpolypeptide may be purified from a native cell, or host cell in the caseof recombinantly produced natural amino acid polypeptides or non-naturalamino acid polypeptides. By way of example a preparation of a naturalamino acid polypeptide or a non-natural amino acid polypeptide may be“substantially purified” when the preparation contains less than about30%, less than about 25%, less than about 20%, less than about 15%, lessthan about 10%, less than about 5%, less than about 4%, less than about3%, less than about 2%, or less than about 1% (by dry weight) ofcontaminating material. By way of example when a natural amino acidpolypeptide or a non-natural amino acid polypeptide is recombinantlyproduced by host cells, the natural amino acid polypeptide ornon-natural amino acid polypeptide may be present at about 30%, about25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%,about 2%, or about 1% or less of the dry weight of the cells. By way ofexample when a natural amino acid polypeptide or a non-natural aminoacid polypeptide is recombinantly produced by host cells, the naturalamino acid polypeptide or non-natural amino acid polypeptide may bepresent in the culture medium at about 5 g/L, about 4 g/L, about 3 g/L,about 2 g/L, about 1 g/L, about 750 mg/L, about 500 mg/L, about 250mg/L, about 100 mg/L, about 50 mg/L, about 10 mg/L, or about 1 mg/L orless of the dry weight of the cells. By way of example, “substantiallypurified” natural amino acid polypeptides or non-natural amino acidpolypeptides may have a purity level of about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater asdetermined by appropriate methods, including, but not limited to,SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

The term “substituents” also referred to as “non-interferingsubstituents” “refers to groups which may be used to replace anothergroup on a molecule. Such groups include, but are not limited to, halo,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkoxy, C₅-C₁₂aralkyl, C₃-C₁₂ cycloalkyl, C₄-C₁₂ cycloalkenyl, phenyl, substitutedphenyl, toluolyl, xylenyl, biphenyl, C₂-C₁₂ alkoxyalkyl, C₅-C₁₂alkoxyaryl, C₅-C₁₂ aryloxyalkyl, C₇-C₁₂ oxyaryl, C₁-C₆ alkylsulfinyl,C₁-C₁₀alkylsulfonyl, —(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein m is from 1 to8, aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclicradical, substituted heterocyclic radical, nitroalkyl, —NO₂, —CN,—NRC(O)—(C₁-C₁₀ alkyl), —C(O)—(C₁-C₁₀ alkyl), C₂-C₁₀ alkthioalkyl,—C(O)O—(C₁-C₁₀ alkyl), —OH, —SO₂, ═S, —COOH, —NR₂, carbonyl,—C(O)—(C₁-C₁₀ alkyl)-CF₃, —C(O)—CF₃, —C(O)NR₂, —(C₁-C₁₀ aryl)-S—(C₆-C₁₀aryl), —C(O)—(C₆-C₁₀ aryl), —(CH₂)_(m)—O—(CH₂)_(m)—O—(C₁-C₁₀ alkyl)wherein each m is from 1 to 8, —C(O)NR₂, —C(S)NR₂, —SO₂NR₂, —NRC(O)NR₂,—NRC(S)NR₂, salts thereof, and the like. Each R group in the precedinglist includes, but is not limited to, H, alkyl or substituted alkyl,aryl or substituted aryl, or alkaryl. Where substituent groups arespecified by their conventional chemical formulas, written from left toright, they equally encompass the chemically identical substituents thatwould result from writing the structure from right to left; for example,—CH₂O— is equivalent to —OCH₂—.

By way of example only, substituents for alkyl and heteroalkyl radicals(including those groups referred to as alkylene, alkenyl,heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl,cycloalkenyl, and heterocycloalkenyl) includes, but is not limited to:—OR, ═O, ═NR, ═N—OR, —NR₂, —SR, -halogen, —SiR₃, —OC(O)R, —C(O)R, —CO₂R,—CONR₂, —OC(O)NR₂, —NRC(O)R, —NRC(O)NR₂, —NR(O)₂R, —NR—C(NR₂)═NR,—S(O)R, —S(O)₂R, —S(O)₂NR₂, —NRSO₂R, —CN and —NO₂. Each R group in thepreceding list includes, but is not limited to, hydrogen, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl, includingbut not limited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or aralkyl groups.When two R groups are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR₂ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl.

By way of example, substituents for aryl and heteroaryl groups include,but are not limited to, —OR, ═O, ═NR, ═N—OR, —NR₂, —SR, -halogen, —SiR₃,—OC(O)R, —C(O)R, —CO₂R, —CONR₂, —OC(O)NR₂, —NRC(O)R, —NRC(O)NR₂,—NR(O)₂R, —NR—C(NR₂)═NR, —S(O)R, —S(O)₂R, —S(O)₂NR₂, —NRSO₂R, —CN, —NO₂,—R, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, in anumber ranging from zero to the total number of open valences on thearomatic ring system; and where each R group in the preceding listincludes, but is not limited to, hydrogen, alkyl, heteroalkyl, aryl andheteroaryl.

The term “therapeutically effective amount,” as used herein, refers tothe amount of a composition containing at least one non-natural aminoacid polypeptide and/or at least one modified non-natural amino acidpolypeptide administered to a patient already suffering from a disease,condition or disorder, sufficient to cure or at least partially arrest,or relieve to some extent one or more of the symptoms of the disease,disorder or condition being treated. The effectiveness of suchcompositions depend conditions including, but not limited to, theseverity and course of the disease, disorder or condition, previoustherapy, the patient's health status and response to the drugs, and thejudgment of the treating physician. By way of example only,therapeutically effective amounts may be determined by routineexperimentation, including but not limited to a dose escalation clinicaltrial.

The term “thioalkoxy,” as used herein, refers to sulfur containing alkylgroups linked to molecules via an oxygen atom.

The term “thermal melting point” or Tm is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of probescomplementary to a target hybridize to the target sequence atequilibrium.

The term “toxic moiety” or “toxic group” as used herein, refers to acompound which can cause harm, disturbances, or death. Toxic moietiesinclude, but are not limited to, NCA1, auristatin, DNA minor groovebinding agent, DNA minor groove alkylating agent, enediyne, lexitropsin,duocarmycin, taxane, puromycin, dolastatin, maytansinoid, vincaalkaloid, AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel,docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin,cyanomorpholino-doxorubicin, dolastatin-10, echinomycin,combretatstatin, chalicheamicin, maytansine, DM-1, netropsin,podophyllotoxin (e.g. etoposide, teniposide, etc.), baccatin and itsderivatives, anti-tubulin agents, cryptophysin, combretastatin,auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16,camptothecin, epothilone A, epothilone B, nocodazole, colchicines,colcimid, estramustine, cemadotin, discodermolide, maytansine,eleutherobin, mechlorethamine, cyclophosphamide, melphalan, carmustine,lomustine, semustine, streptozocin, chlorozotocin, uracil mustard,chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine,triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide,ytarabine, cytosine arabinoside, fluorouracil, floxuridine,6-thioguanine, 6-mercaptopurine, pentostatin, 5-fluorouracil,methotrexate, 10-propargyl-5,8-dideazafolate, 5,8-dideazatetrahydrofolicacid, leucovorin, fludarabine phosphate, pentostatine, gemcitabine,Ara-C, paclitaxel, docetaxel, deoxycoformycin, mitomycin-C,L-asparaginase, azathioprine, brequinar, antibiotics (e.g.,anthracycline, gentamicin, cefalotin, vancomycin, telavancin,daptomycin, azithromycin, erythromycin, rocithromycin, furazolidone,amoxicillin, ampicillin, carbenicillin, flucloxacillin, methicillin,penicillin, ciprofloxacin, moxifloxacin, ofloxacin, doxycycline,minocycline, oxytetracycline, tetracycline, streptomycin, rifabutin,ethambutol, rifaximin, etc.), antiviral drugs (e.g., abacavir,acyclovir, ampligen, cidofovir, delavirdine, didanosine, efavirenz,entecavir, fosfonet, ganciclovir, ibacitabine, immunovir, idoxuridine,inosine, lopinavir, methisazone, nexavir, nevirapine, oseltamivir,penciclovir, stavudine, trifluridine, truvada, valaciclovir, zanamivir,etc.), daunorubicin hydrochloride, daunoriycin, rubidomycin, cerubidine,idarubicin, doxorubicin, epirubicin and morpholino derivatives,phenoxizone biscyclopeptides (e.g., dactinomycin), basic glycopeptides(e.g., bleomycin), anthraquinone glycosides (e.g., plicamycin,mithramycin), anthracenediones (e.g., mitoxantrone), azirinopyrroloindolediones (e.g., mitomycin), macrocyclic immunosuppressants (e.g.,cyclosporine, FK-506, tacrolimus, prograf, rapamycin etc.), navelbene,CPT-11, anastrazole, letrazole, capecitabine, reloxafine,cyclophosphamide, ifosamide, droloxafine, allocolchicine, HalichondrinB, colchicine, colchicine derivatives, maytansine, rhizoxin, paclitaxel,paclitaxel derivatives, docetaxel, thiocolchicine, trityl cysterin,vinblastine sulfate, vincristine sulfate, cisplatin, carboplatin,hydroxyurea, N-methylhydrazine, epidophyllotoxin, procarbazine,mitoxantrone, leucovorin, and tegafur. “Taxanes” include paclitaxel, aswell as any active taxane derivative or pro-drug. Chemotherapeuticagents such as erlotinib (TARCEVA®, Genentech/OSI Pharm.), bortezomib(VELCADE®, Millenium Pharm.), fulvestrant (FASLODEX®, AstraZeneca),sutent (SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), PTK787/ZK 222584 (Novartis), oxaliplatin(Eloxatin®, Sanofi), 5-FU (5-fluorouracil), leucovorin, Rapamycin(Sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016,GlaxoSmithKline), lonafarnib (SCH 66336), sorafenib (BAY43-9006, BayerLabs.), and gefitinib (IRESSA®, AstraZeneca), AG1478, AG1571 (SU 5271;Sugen), alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; antifolate antineoplastic such as pemetrexed (ALIMTA® EliLilly), aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics,calicheamicin, calicheamicin gamma1I and calicheamicin omegaI1;dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores, aclacinomysins,actinomycin, anthramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.,paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.),ABRAXANE™ Cremophor-free, albumin, nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

The terms “treat,” “treating” or “treatment”, as used herein, includealleviating, abating or ameliorating a disease or condition symptoms,preventing additional symptoms, ameliorating or preventing theunderlying metabolic causes of symptoms, inhibiting the disease orcondition, e.g., arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or to condition. The terms “treat,”“treating” or “treatment”, include, but are not limited to, prophylacticand/or therapeutic treatments.

As used herein, the term “water soluble polymer” refers to any polymerthat is soluble in aqueous solvents. Such water soluble polymersinclude, but are not limited to, polyethylene glycol, polyethyleneglycol propionaldehyde, mono C₁-C₁₀ alkoxy or aryloxy derivativesthereof (described in U.S. Pat. No. 5,252,714 which is incorporated byreference herein), monomethoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinylether maleicanhydride, N-(2-Hydroxypropyl)-methacrylamide, dextran, dextranderivatives including dextran sulfate, polypropylene glycol,polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol,heparin, heparin fragments, polysaccharides, oligosaccharides, glycans,cellulose and cellulose derivatives, including but not limited tomethylcellulose and carboxymethyl cellulose, serum albumin, starch andstarch derivatives, polypeptides, polyalkylene glycol and derivativesthereof, copolymers of polyalkylene glycols and derivatives thereof,polyvinyl ethyl ethers, andalpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, ormixtures thereof. By way of example only, coupling of such water solublepolymers to natural amino acid polypeptides or non-natural polypeptidesmay result in changes including, but not limited to, increased watersolubility, increased or modulated serum half-life, increased ormodulated therapeutic half-life relative to the unmodified form,increased bioavailability, modulated biological activity, extendedcirculation time, modulated immunogenicity, modulated physicalassociation characteristics including, but not limited to, aggregationand multimer formation, altered receptor binding, altered binding to oneor more binding partners, and altered receptor dimerization ormultimerization. In addition, such water soluble polymers may or may nothave their own biological activity.

Unless otherwise indicated, conventional methods of mass spectroscopy,NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniquesand pharmacology, within the skill of the art are employed.

Compounds, (including, but not limited to non-natural amino acids,non-natural amino acid polypeptides, modified non-natural amino acidpolypeptides, and reagents for producing the aforementioned compounds)presented herein include isotopically-labeled compounds, which areidentical to those recited in the various formulas and structurespresented herein, but for the fact that one or more atoms are replacedby an atom having an atomic mass or mass number different from theatomic mass or mass number usually found in nature. Examples of isotopesthat can be incorporated into the present compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as ²H,³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F, ³⁶Cl, respectively. Certainisotopically-labeled compounds described herein, for example those intowhich radioactive isotopes such as ³H and ¹⁴C are incorporated, areuseful in drug and/or substrate tissue distribution assays. Further,substitution with isotopes such as deuterium, i.e., ²H, can affordcertain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements.

Some of the compounds herein (including, but not limited to non-naturalamino acids, non-natural amino acid polypeptides and modifiednon-natural amino acid polypeptides, and reagents for producing theaforementioned compounds) have asymmetric carbon atoms and can thereforeexist as enantiomers or diastereomers. Diasteromeric mixtures can beseparated into their individual diastereomers on the basis of theirphysical chemical differences by methods known, for example, bychromatography and/or fractional crystallization. Enantiomers can beseparated by converting the enantiomeric mixture into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,alcohol), separating the diastereomers and converting (e.g.,hydrolyzing) the individual diastereomers to the corresponding pureenantiomers. All such isomers, including diastereomers, enantiomers, andmixtures thereof are considered as part of the compositions describedherein.

In additional or further embodiments, the compounds described herein(including, but not limited to non-natural amino acids, non-naturalamino acid polypeptides and modified non-natural amino acidpolypeptides, and reagents for producing the aforementioned compounds)are used in the form of pro-drugs. In additional or further embodiments,the compounds described herein ((including, but not limited tonon-natural amino acids, non-natural amino acid polypeptides andmodified non-natural amino acid polypeptides, and reagents for producingthe aforementioned compounds) are metabolized upon administration to anorganism in need to produce a metabolite that is then used to produce adesired effect, including a desired therapeutic effect. In further oradditional embodiments are active metabolites of non-natural amino acidsand “modified or unmodified” non-natural amino acid polypeptides.

The methods and formulations described herein include the use ofN-oxides, crystalline forms (also known as polymorphs), orpharmaceutically acceptable salts of non-natural amino acids,non-natural amino acid polypeptides and modified non-natural amino acidpolypeptides. In certain embodiments, non-natural amino acids,non-natural amino acid polypeptides and modified non-natural amino acidpolypeptides may exist as tautomers. All tautomers are included withinthe scope of the non-natural amino acids, non-natural amino acidpolypeptides and modified non-natural amino acid polypeptides presentedherein. In addition, the non-natural amino acids, non-natural amino acidpolypeptides and modified non-natural amino acid polypeptides describedherein can exist in unsolvated as well as solvated forms withpharmaceutically acceptable solvents such as water, ethanol, and thelike. The solvated forms of the non-natural amino acids, non-naturalamino acid polypeptides and modified non-natural amino acid polypeptidespresented herein are also considered to be disclosed herein.

Some of the compounds herein (including, but not limited to non-naturalamino acids, non-natural amino acid polypeptides and modifiednon-natural amino acid polypeptides and reagents for producing theaforementioned compounds) may exist in several tautomeric forms. Allsuch tautomeric forms are considered as part of the compositionsdescribed herein. Also, for example all enol-keto forms of any compounds(including, but not limited to non-natural amino acids, non-naturalamino acid polypeptides and modified non-natural amino acid polypeptidesand reagents for producing the aforementioned compounds) herein areconsidered as part of the compositions described herein.

Some of the compounds herein (including, but not limited to non-naturalamino acids, non-natural amino acid polypeptides and modifiednon-natural amino acid polypeptides and reagents for producing either ofthe aforementioned compounds) are acidic and may form a salt with apharmaceutically acceptable cation. Some of the compounds herein(including, but not limited to non-natural amino acids, non-naturalamino acid polypeptides and modified non-natural amino acid polypeptidesand reagents for producing the aforementioned compounds) can be basicand accordingly, may form a salt with a pharmaceutically acceptableanion. All such salts, including di-salts are within the scope of thecompositions described herein and they can be prepared by conventionalmethods. For example, salts can be prepared by contacting the acidic andbasic entities, in either an aqueous, non-aqueous or partially aqueousmedium. The salts are recovered by using at least one of the followingtechniques: filtration, precipitation with a non-solvent followed byfiltration, evaporation of the solvent, or, in the case of aqueoussolutions, lyophilization.

Pharmaceutically acceptable salts of the non-natural amino acidpolypeptides disclosed herein may be formed when an acidic protonpresent in the parent non-natural amino acid polypeptides either isreplaced by a metal ion, by way of example an alkali metal ion, analkaline earth ion, or an aluminum ion; or coordinates with an organicbase. In addition, the salt forms of the disclosed non-natural aminoacid polypeptides can be prepared using salts of the starting materialsor intermediates. The non-natural amino acid polypeptides describedherein may be prepared as a pharmaceutically acceptable acid additionsalt (which is a type of a pharmaceutically acceptable salt) by reactingthe free base form of non-natural amino acid polypeptides describedherein with a pharmaceutically acceptable inorganic or organic acid.Alternatively, the non-natural amino acid polypeptides described hereinmay be prepared as pharmaceutically acceptable base addition salts(which are a type of a pharmaceutically acceptable salt) by reacting thefree acid form of non-natural amino acid polypeptides described hereinwith a pharmaceutically acceptable inorganic or organic base.

The type of pharmaceutical acceptable salts, include, but are notlimited to: (1) acid addition salts, formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with organic acids such asacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,2-naphthalenesulfonic acid,4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like; (2) salts formed when anacidic proton present in the parent compound either is replaced by ametal ion, e.g., an alkali metal ion, an alkaline earth ion, or analuminum ion; or coordinates with an organic base. Acceptable organicbases include ethanolamine, diethanolamine, triethanolamine,tromethamine, N-methylglucamine, and the like. Acceptable inorganicbases include aluminum hydroxide, calcium hydroxide, potassiumhydroxide, sodium carbonate, sodium hydroxide, and the like.

The corresponding counterions of the non-natural amino acid polypeptidepharmaceutical acceptable salts may be analyzed and identified usingvarious methods including, but not limited to, ion exchangechromatography, ion chromatography, capillary electrophoresis,inductively coupled plasma, atomic absorption spectroscopy, massspectrometry, or any combination thereof. In addition, the therapeuticactivity of such non-natural amino acid polypeptide pharmaceuticalacceptable salts may be tested using the techniques and methodsdescribed in examples 87-91.

It should be understood that a reference to a salt includes the solventaddition forms or crystal forms thereof, particularly solvates orpolymorphs. Solvates contain either stoichiometric or non-stoichiometricamounts of a solvent, and are often formed during the process ofcrystallization with pharmaceutically acceptable solvents such as water,ethanol, and the like. Hydrates are formed when the solvent is water, oralcoholates are formed when the solvent is alcohol. Polymorphs includethe different crystal packing arrangements of the same elementalcomposition of a compound. Polymorphs usually have different X-raydiffraction patterns, infrared spectra, melting points, density,hardness, crystal shape, optical and electrical properties, stability,and solubility. Various factors such as the recrystallization solvent,rate of crystallization, and storage temperature may cause a singlecrystal form to dominate.

The screening and characterization of non-natural amino acid polypeptidepharmaceutical acceptable salts polymorphs and/or solvates may beaccomplished using a variety of techniques including, but not limitedto, thermal analysis, x-ray diffraction, spectroscopy, vapor sorption,and microscopy. Thermal analysis methods address thermo chemicaldegradation or thermo physical processes including, but not limited to,polymorphic transitions, and such methods are used to analyze therelationships between polymorphic forms, determine weight loss, to findthe glass transition temperature, or for excipient compatibilitystudies. Such methods include, but are not limited to, Differentialscanning calorimetry (DSC), Modulated Differential Scanning Calorimetry(MDCS), Thermogravimetric analysis (TGA), and Thermogravi-metric andInfrared analysis (TG/IR). X-ray diffraction methods include, but arenot limited to, single crystal and powder diffractometers andsynchrotron sources. The various spectroscopic techniques used include,but are not limited to, Raman, FTIR, UVIS, and NMR (liquid and solidstate). The various microscopy techniques include, but are not limitedto, polarized light microscopy, Scanning Electron Microscopy (SEM) withEnergy Dispersive X-Ray Analysis (EDX), Environmental Scanning ElectronMicroscopy with EDX (in gas or water vapor atmosphere), IR microscopy,and Raman microscopy.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 presents a graphical illustration of anti-tumor efficacy ofunconjugated ARX-αCD70 antibody CD70-2H5-HA119 administered in a singledose to female nude mice 7 days following implantation of a 786-O tumorfragment in a dose of 3 mg/kg) against control vehicle.

FIG. 2 presents a graphical illustration of tumor volume in female nudemice test subject groups treated with 1 mg/kg, 3 mg/kg, and 10 mg/kgdoses of the antibody drug conjugate CD70-2H5-HA119-NCA1 in the renalcell carcinoma xenograft model, same conditions from FIG. 1.

FIG. 3A presents a graphical illustration of the results from acytotoxicity assay of three ARX-antibody drug conjugates; CD70-2H5-NCA1,CD70-69A7-NCA1, and CD70hIF6-NCA1 in 786-O cells.

FIG. 3B presents a graphical illustration of the results from acytotoxicity assay of three ARX-antibody drug conjugates; CD70-2H5-NCA1,CD70-69A7-NCA1, and CD70hIF6-NCA1 in Caki-1 cells.

FIG. 4 presents a graphical illustration of tumor volume over timefollowing a single injection at day 12 of FIG. 4(A) αCD70 antibodyCD70-2H5-HA119 conjugated to AS269 (also referred to as NCA1 within thepresent application); FIG. 4(B) αCD70 antibody CD70-8A1-HA122 conjugatedto AS269; and FIG. 4(C) αCD70 antibody CD70-7F2-HA119 conjugated. Theantibody drug conjugates were given in doses of 0.5 mg/kg; 1 mg/kg; and3 mg/kg and compared to vehicle.

FIG. 5 presents a dot plot of the tumor volumes from the different testgroups as measured on day 49 following transplantation of 786-O tumorfragment.

FIG. 6A presents a graphical illustration of tumor volume in female nudemice test subject groups treated with CD70-2H5-AS269 3 mg/kg;CD70-8A1-AS269 3 mg/kg; and CD70-7F2-AS269 3 mg/kg; administered 12 daysfollowing transplantation of a 786-O tumor fragment.

FIG. 6B presents a graphical illustration of tumor volume in female nudemice test subject groups treated with CD70-2H5-AS269 in 1 mg/kg and 3mg/kg as compared to vehicle; antibody drug conjugates were administered12 days following transplantation of a 786-O tumor fragment.

FIG. 6C presents a graphical illustration of tumor volume in female nudemice test subject groups treated with 8A1-pAF1-AS269 in 1 mg/kg and 3mg/kg as compared to vehicle; antibody drug conjugates were administered12 days following transplantation of a 786-O tumor fragment.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

I. Introduction

Recently, an entirely new technology in the protein sciences has beenreported, which promises to overcome many of the limitations associatedwith site-specific modifications of proteins. Specifically, newcomponents have been added to the protein biosynthetic machinery of theprokaryote Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001),Science 292:498-500) and the eukaryote Sacchromyces cerevisiae (S.cerevisiae) (e.g., J. Chin et al., Science 301:964-7 (2003)), which hasenabled the incorporation of non-natural amino acids to proteins invivo. A number of new amino acids with novel chemical, physical orbiological properties, including photoaffinity labels andphotoisomerizable amino acids, keto amino acids, and glycosylated aminoacids have been incorporated efficiently and with high fidelity intoproteins in E. coli and in yeast in response to the amber codon, TAG,using this methodology. See, e.g., J. W. Chin et al., (2002), Journal ofthe American Chemical Society 124:9026-9027 (incorporated by referencein its entirety); J. W. Chin, & P. G. Schultz, (2002), ChemBioChem3(11):1135-1137 (incorporated by reference in its entirety); J. W. Chin,et al., (2002), PNAS United States of America 99(17):11020-11024(incorporated by reference in its entirety); and, L. Wang, & P. G.Schultz, (2002), Chem. Comm., 1-11 (incorporated by reference in itsentirety). These studies have demonstrated that it is possible toselectively and routinely introduce chemical functional groups that arenot found in proteins, that are chemically inert to all of thefunctional groups found in the 20 common, genetically-encoded aminoacids and that may be used to react efficiently and selectively to formstable covalent linkages.

II. Overview

The present invention provides αCD70 antibodies having a non-naturallyencoded amino acid that facilitate antibody conjugation to a drug (e.g.a drug, toxin, marker molecule). In one embodiment, the ADC comprise anαCD70 antibody conjugated to a drug wherein the conjugation occurs via anon-naturally encoded amino acid in the antibody. In one embodiment, theADC comprise an αCD70 antibody conjugated to a drug wherein theconjugation occurs via a non-naturally encoded amino acid in the heavychain of the antibody. In one embodiment, the ADC comprise an αCD70antibody conjugated to a drug wherein the conjugation occurs via anon-naturally encoded amino acid in the light chain of the antibody. Inone embodiment, the ADC comprise a full-length antibody conjugated to adrug wherein the conjugation occurs via a non-naturally encoded aminoacid in the antibody.

In one embodiment, the ADC comprise a full-length antibody conjugated toa drug wherein the conjugation occurs via a non-naturally encoded aminoacid in the heavy chain of the antibody. In one embodiment, the ADCcomprise a full-length antibody conjugated to a drug wherein theconjugation occurs via a non-naturally encoded amino acid in the lightchain of the antibody.

In some embodiments, the drug of the ADC is a cytotoxic drug. In someaspects of the invention, the cytotoxic drug is selected from the groupconsisting of an auristatin, a DNA minor groove binding agent, a DNAminor groove alkylating agent, an enediyne, a lexitropsin, aduocarmycin, a taxane, a puromycin, a dolastatin, a maytansinoid, and avinca alkaloid. In some aspects of the invention, the cytotoxic drug is

In other embodiments of the present invention, the cytotoxic drug ischosen from the group comprising:

In some embodiments of the present invention, the cytotoxic drug ischosen from the group consisting of

In other aspects of the present invention, the cytotoxic drug is chosenfrom the group consisting of:

In other aspects of the present invention, the cytotoxic drug is chosenfrom the group consisting of

In other aspects of the present invention, the cytotoxic drug is chosenfrom the group consisting of AFP, MMAF, MMAE, AEB, AEVB, auristatin E,paclitaxel, docetaxel, CC-1065, SN-38, topotecan,morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin,dolastatin-10, echinomycin, combretatstatin, chalicheamicin, maytansine,DM-1, or netropsin.

In some aspects of the invention, the cytotoxic drug is an anti-tubulinagent. In some embodiments, the anti-tubulin agent is an auristatin, avinca alkaloid, a podophyllotoxin, a taxane, a baccatin derivative, acryptophysin, a maytansinoid, a combretastatin, or a dolastatin. Inother aspects of the invention, the antitubulin agent is AFP, MMAF,MMAE, AEB, AEVB, auristatin E, vincristine, vinblastine, vindesine,vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A,epothilone B, nocodazole, colchicines, colcimid, estramustine,cemadotin, discodennolide, maytansine, DM-1, or eleutherobin.

In other aspects of the invention, the cytotoxic drug of the ADC isgancyclovir, etanercept, cyclosporine, tacrolimus, rapamycin,cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate,cortisol, aldosterone, dexamethasone, a cyclooxygenase inhibitor, a5-ipoxygenase inhibitor, or a leukotriene receptor antagonist.

In some embodiments of the invention, the antibody of the ADC comprisesa full length antibody that: (a) binds to CD70, and (b) is conjugated toa cytotoxic agent or an immunosuppressive agent, wherein theantibody-drug conjugate exerts: (a) a cytotoxic or cytostatic effect ona CD70-expressing cancer cell line, or (b) a cytotoxic, cytostatic, orimmunosuppressive effect on a CD70-expressing immune cell, wherein theconjugation occurs at a non-naturally encoded amino acid in theantibody.

At one level, described herein are the tools (methods, compositions,techniques) for creating and using dolastatin linker derivatives oranalogs comprising at least one carbonyl, dicarbonyl, oxime,hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone,thioester, ester, dicarbonyl, hydrazine, azide, amidine, imine, diamine,keto-amine, keto-alkyne, alkyne, cycloalkyne, or ene-dione. At anotherlevel, described herein are the tools (methods, compositions,techniques) for creating and using dolastatin linker derivatives oranalogs comprising at least one non-natural amino acid or modifiednon-natural amino acid with an oxime, aromatic amine, heterocycle (e.g.,indole, quinoxaline, phenazine, pyrazole, triazole, etc.).

Such dolastatin linker derivatives comprising non-natural amino acidsmay contain further functionality, including but not limited to, apolymer; a water-soluble polymer; a derivative of polyethylene glycol; asecond protein or polypeptide or polypeptide analog; an antibody orantibody fragment; and any combination thereof. Note that the variousaforementioned functionalities are not meant to imply that the membersof one functionality cannot be classified as members of anotherfunctionality. Indeed, there will be overlap depending upon theparticular circumstances. By way of example only, a water-solublepolymer overlaps in scope with a derivative of polyethylene glycol,however the overlap is not complete and thus both functionalities arecited above.

Provided herein in some embodiments, is a toxic group linker derivativecomprising a carbonyl, dicarbonyl, oxime, hydroxylamine, aldehyde,protected aldehyde, ketone, protected ketone, thioester, ester,dicarbonyl, hydrazine, azide, amidine, imine, diamine, keto-amine,keto-alkyne, alkyne, cycloalkyne, or ene-dione. In some embodiments, thetoxic group derivative comprises any of the linkers disclosed herein. Inother embodiments, described herein are the tools (methods,compositions, techniques) for creating and using toxic group derivativesor analogs comprising at least one non-natural amino acid or modifiednon-natural amino acid with an oxime, aromatic amine, heterocycle (e.g.,indole, quinoxaline, phenazine, pyrazole, triazole, etc.).

In some embodiments, such toxic derivatives comprising non-natural aminoacids may contain further functionality, including but not limited to, apolymer; a water-soluble polymer; a derivative of polyethylene glycol; asecond protein or polypeptide or polypeptide analog; an antibody orantibody fragment; and any combination thereof. In specific embodiments,the toxic group is a tubulin inhibitor. In certain specific embodiments,the toxic group is dolastatin or auristatin. In other specificembodiments, the toxic group is dolastatin or auristatin derivative.Note that the various aforementioned functionalities are not meant toimply that the members of one functionality cannot be classified asmembers of another functionality. Indeed, there will be overlapdepending upon the particular circumstances. By way of example only, awater-soluble polymer overlaps in scope with a derivative ofpolyethylene glycol, however the overlap is not complete and thus bothfunctionalities are cited above.

Certain embodiments of the present invention describe preparations ofcertain toxic moieties with linkers that reduce the toxicity of themoiety in vivo while the toxic moiety retains pharmacological activity.In some embodiments, the toxicity of the linked toxic group, whenadministered to an animal or human, is reduced or eliminated compared tothe free toxic group or toxic group derivatives comprising labilelinkages, while retaining pharmacological activity. In some embodiments,increased doses of the linked toxic group (e.g., dolastatin linkerderivatives, non-natural amino acid linked dolastatin derivatives) maybe administered to animals or humans with greater safety. In certainembodiments, the non-natural amino acid polypeptides linked to a toxicmoiety (e.g., dolastatin derivative) provides in vitro and in vivostability. In some embodiments, the non-natural amino acid polypeptideslinked to a toxic moiety (e.g., tubulin inhibitor, dolastatin-10derivative) are efficacious and less toxic compared to the free toxicmoiety (e.g., tubulin inhibitor, dolastatin-10).

III. Dolastatin Linker Derivatives

At one level, described herein are the tools (methods, compositions,techniques) for creating and using a dolastatin linker derivatives oranalogs comprising at least one non-natural amino acid or modifiednon-natural amino acid with a carbonyl, dicarbonyl, oxime orhydroxylamine group. Such dolastatin linker derivatives comprisingnon-natural amino acids may contain further functionality, including butnot limited to, a polymer; a water-soluble polymer; a derivative ofpolyethylene glycol; a second protein or polypeptide or polypeptideanalog; an antibody or antibody fragment; and any combination thereof.Note that the various aforementioned functionalities are not meant toimply that the members of one functionality cannot be classified asmembers of another functionality. Indeed, there will be overlapdepending upon the particular circumstances. By way of example only, awater-soluble polymer overlaps in scope with a derivative ofpolyethylene glycol, however the overlap is not complete and thus bothfunctionalities are cited above.

In one aspect are methods for selecting and designing a dolastatinlinker derivative to be modified using the methods, compositions andtechniques described herein. The new dolastatin linker derivative may bedesigned de novo, including by way of example only, as part ofhigh-throughput screening process (in which case numerous polypeptidesmay be designed, synthesized, characterized and/or tested) or based onthe interests of the researcher. The new dolastatin linker derivativemay also be designed based on the structure of a known or partiallycharacterized polypeptide. By way of example only, dolastatin has beenthe subject of intense study by the scientific community; a new compoundmay be designed based on the structure of dolastatin. The principles forselecting which amino acid(s) to substitute and/or modify are describedseparately herein. The choice of which modification to employ is alsodescribed herein, and can be used to meet the need of the experimenteror end user. Such needs may include, but are not limited to,manipulating the therapeutic effectiveness of the polypeptide, improvingthe safety profile of the polypeptide, adjusting the pharmacokinetics,pharmacologics and/or pharmacodynamics of the polypeptide, such as, byway of example only, increasing water solubility, bioavailability,increasing serum half-life, increasing therapeutic half-life, modulatingimmunogenicity, modulating biological activity, or extending thecirculation time. In addition, such modifications include, by way ofexample only, providing additional functionality to the polypeptide,incorporating an antibody, and any combination of the aforementionedmodifications.

Also described herein are dolastatin linker derivatives that have or canbe modified to contain an oxime, carbonyl, dicarbonyl, or hydroxylaminegroup. Included with this aspect are methods for producing, purifying,characterizing and using such dolastatin linker derivatives

The dolastatin linker derivative may contain at least one, at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, or ten or more of a carbonyl ordicarbonyl group, oxime group, hydroxylamine group, or protected formsthereof. The dolastatin linker derivative can be the same or different,for example, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, or more different sites in the derivative thatcomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or more different reactive groups.

A. Structure and Synthesis of Dolastatin Linker Derivatives:Electrophilc and Nucleophilic Groups

Dolastatin derivatives with linkers containing a hydroxylamine (alsocalled an aminooxy) group allow for reaction with a variety ofelectrophilic groups to form conjugates (including but not limited to,with PEG or other water soluble polymers). Like hydrazines, hydrazidesand semicarbazides, the enhanced nucleophilicity of the aminooxy grouppermits it to react efficiently and selectively with a variety ofmolecules that contain carbonyl- or dicarbonyl-groups, including but notlimited to, ketones, aldehydes or other functional groups with similarchemical reactivity. See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc.117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34(9):727-736 (2001). Whereas the result of reaction with a hydrazine group isthe corresponding hydrazone, however, an oxime results generally fromthe reaction of an aminooxy group with a carbonyl- ordicarbonyl-containing group such as, by way of example, a ketones,aldehydes or other functional groups with similar chemical reactivity.In some embodiments, dolastatin derivatives with linkers comprising anazide, alkyne or cycloalkyne allow for linking of molecules viacycloaddition reactions (e.g., 1,3-dipolar cycloadditions, azide-alkyneHuisgen cycloaddition, etc.). (Described in U.S. Pat. No. 7,807,619which is incorporated by reference herein to the extent relative to thereaction).

Thus, in certain embodiments described herein are dolastatin derivativeswith linkers comprising a hydroxylamine, aldehyde, protected aldehyde,ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine,amidine, imine, diamine, keto-amine, keto-alkyne, and ene-dionehydroxylamine group, a hydroxylamine-like group (which has reactivitysimilar to a hydroxylamine group and is structurally similar to ahydroxylamine group), a masked hydroxylamine group (which can be readilyconverted into a hydroxylamine group), or a protected hydroxylaminegroup (which has reactivity similar to a hydroxylamine group upondeprotection). In some embodiments, the dolastatin derivatives withlinkers comprise azides, alkynes or cycloalkynes. Such dolastatin linkerderivatives include compounds having the structure of Formula (I),(III), (IV), (V), and (VI):

wherein:

-   -   Z has the structure of:

-   -   -   R₅ is H, COR₈, C₁-C₆alkyl, or thiazole;            -   R₈ is OH or —NH-(alkylene-O)_(n)—NH₂;        -   R₆ is OH or H;        -   Ar is phenyl or pyridine;

    -   R₇ is C₁-C₆alkyl or hydrogen;

    -   Y and V are each selected from the group consisting of an        hydroxylamine, methyl, aldehyde, protected aldehyde, ketone,        protected ketone, thioester, ester, dicarbonyl, hydrazine,        azide, amidine, imine, diamine, keto-amine, keto-alkyne, alkyne,        cycloalkyne, and ene-dione;

    -   L, L₁, L₂, L₃, and L₄ are each linkers selected from the group        consisting of a bond, -alkylene-, -alkylene-C(O)—, -alkylene-J-,        -(alkylene-O)_(n)-alkylene-, -(alkylene-O)_(n)-alkylene-C(O)—,        -(alkylene-O)_(n)-J-, -(alkylene-O)_(n)-J-alkylene-,        -(alkylene-O)_(n)—(CH₂)_(n′)—NHC(O)—(CH₂)_(n″)—C(Me)₂-S—S—(CH₂)_(n′″)—NHC(O)-(alkylene-O)_(n″″)-alkylene-,        -(alkylene-O)_(n)-alkylene-W—, -alkylene-C(O)—W—,        -(alkylene-O)_(n)-alkylene-J-,        -alkylene′-J-(alkylene-O)_(n)-alkylene-,        -(alkylene-O)_(n)-alkylene-J-alkylene′,        -J-(alkylene-O)_(n)-alkylene-,        -(alkylene-O)_(n)-alkylene-J-(alkylene-O)_(n)′-alkylene-J′-,        —W—, -alkylene-W—, alkylene′-J-(alkylene-NMe)_(n)-alkylene-W—,        -J-(alkylene-NMe)_(n)-alkylene-W—,        -(alkylene-O)_(n)-alkylene-U-alkylene-C(O)—,        -(alkylene-O)_(n)-alkylene-U-alkylene-;        -J-alkylene-NMe-alkylene′-NMe-alkylene″-W—, and        -alkylene-J-alylene′-NMe-alkylene″-NMe-alkylene′″-W—;        -   W has the structure of:

-   -   -   U has the structure of:

-   -   each J and J′ independently have the structure of:

-   -   each n, n′n″, n′″ and n″″ are independently integers greater        than or equal to one; and    -   or L is absent, Y is methyl, R₅ is COR₈, and R₈ is        —NH-(alkylene-O)_(n)—NH₂.        Such dolastatin linker derivatives may be in the form of a salt,        or may be incorporated into a non-natural amino acid        polypeptide, polymer, polysaccharide, or a polynucleotide and        optionally post translationally modified.

In certain embodiments of compounds of Formula (I), (III), and (V), R₅is thiazole or carboxylic acid. In certain embodiments of compounds ofFormula (I), (III), and (V), R₅ is hydrogen. In certain embodiments ofcompounds of Formula (I), (III), and (V), R₅ is methyl, ethyl, propyl,iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl.In certain embodiments of compounds of Formula (I), (III), and (V), R₅is —NH-(alkylene-O)_(n)—NH₂, wherein alkylene is —CH₂—, —CH₂CH₂—,—CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,CH₂CH₂CH₂CHzCH₂CH₂CH₂CH₂CH₂CH₂CH—, or—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH—. In certain embodiments ofcompounds of Formula (IV) and (VI), R₅ is —NH-(alkylene-O)_(n)—NH₂,wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In some embodiments, Y is azide. In other embodiments, Y is cycloalkyne.In specific embodiments, the cyclooctyne has a structure of:

-   -   each R₁₉ is independently selected from the group consisting of        C₁-C₆ alkyl, C₁-C₆ alkoxy, ester, ether, thioether, aminoalkyl,        halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl        halide, alkyl amine, alkyl sulfonic acid, alkyl nitro,        thioester, sulfonyl ester, halosulfonyl, nitrile, alkyl nitrile,        and nitro; and    -   q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.

In certain embodiments of compounds of Formula (I), (III), and (V), R₆is H. In some embodiments of compounds of Formula (I), (III), and (V),R₆ is hydroxy.

In certain embodiments of compounds of Formula (I), (III), and (V), Aris phenyl.

In certain embodiments of compounds of Formula (I), (III), (IV), (V),and (VI), R₇ is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyliso-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments ofcompounds of Formula (I), (III), (IV), (V), and (VI), R₇ is hydrogen.

In certain embodiments of compounds of Formula (I), (III), and (V), Y ishydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone,thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine,keto-amine, keto-alkyne, or ene-dione.

In certain embodiments of compounds of Formula (IV) and (VI), V is ahydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protectedketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine,diamine, keto-amine, keto-alkyne, and ene-dione.

In certain embodiments of compounds of Formula (I), (III), (IV), (V),and (VI), each L, L₁, L₂, L₃, and L₄ is independently a cleavable linkeror non-cleavable linker. In certain embodiments of compounds of Formula(I), (III), (IV), (V), and (VI), each L, L₁, L₂, L₃, and L₄ isindependently a oligo(ethylene glycol) derivatized linker.

In certain embodiments of compounds of Formula (I), (III), (IV), (V),and (VI), each alkylene, alkylene′, alkylene″, and alkylene′″independently is —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,or —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—. In certain embodiments ofcompounds of Formula (XIV), (XV), (XVI), (XVII), and (XVIII), each n,n′, n″, n′″, and n″″ is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

B. Structure and Synthesis of Dolastatin Linker Derivatives:Hydroxylamine Groups

Thus, in certain embodiments described herein are dolastatin derivativeswith linkers comprising a hydroxylamine group, a hydroxylamine-likegroup (which has reactivity similar to a hydroxylamine group and isstructurally similar to a hydroxylamine group), a masked hydroxylaminegroup (which can be readily converted into a hydroxylamine group), or aprotected hydroxylamine group (which has reactivity similar to ahydroxylamine group upon deprotection). Such dolastatin linkerderivatives include compounds having the structure of Formula (I):

wherein:

-   -   Z has the structure of:

-   -   -   R₅ is H, COR₈, C₁-C₆alkyl, or thiazole;            -   R₈ is OH or —NH-(alkylene-O)_(n)—NH₂;        -   R₆ is OH or H;        -   Ar is phenyl or pyridine;

    -   R₇ is C₁-C₆alkyl or hydrogen;

    -   Y is NH₂—O— or methyl;

    -   L is a linker selected from the group consisting of alkylene-,        -alkylene-C(O)—, -(alkylene-O)_(n)-alkylene-,        -(alkylene-O)_(n)-alkylene-C(O)—,        -(alkylene-O)_(n)—(CH₂)_(n′)—NHC(O)—(CH₂)_(n″)—CMe)₂-S—S—(CH₂)_(n′″)—NHC(O)-(alkylene-O)_(n″″)-alkylene-,        -(alkylene-O)_(n)-alkylene-W—, -alkylene-C(O)—W—,        -(alkylene-O)_(n)-alkylene-U-alkylene-C(O)—, and        -(alkylene-O)_(n)-alkylene-U-alkylene-;        -   W has the structure of:

-   -   U has the structure of:

-   -   or L is absent, Y is methyl, R₅ is COR₈, and R₈ is        —NH-(alkylene-O)_(n)—NH₂; and each n, n′, n″, n′″ and n″″ are        independently integers greater than or equal to one. Such        dolastatin linker derivatives may be in the form of a salt, or        may be incorporated into a non-natural amino acid polypeptide,        polymer, polysaccharide, or a polynucleotide and optionally post        translationally modified.

In certain embodiments of compounds of Formula (I), R₅ is thiazole. Incertain embodiments of compounds of Formula (I), R₅ is hydrogen. Incertain embodiments of compounds of Formula (I), R₅ is methyl, ethyl,propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, orhexyl. In certain embodiments of compounds of Formula (I), R₅ is—NH-(alkylene-O)_(n)—NH₂, wherein alkylene is —CH₂—, —CH₂CH₂—,—CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, or—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—. In certain embodiments ofcompounds of Formula (I), R₅ is —NH-(alkylene-O)_(n)—NH₂, wherein n is0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments of compounds of Formula (I), R₆ is H. In someembodiments of compounds of Formula (I), R₆ is hydroxy.

In certain embodiments of compounds of Formula (I), Ar is phenyl.

In certain embodiments of compounds of Formula (I), R₇ is methyl, ethyl,propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl, pentyl, orhexyl. In certain embodiments of compounds of Formula (I), R₇ ishydrogen.

In certain embodiments of compounds of Formula (I), Y is hydroxylamine,aldehyde, protected aldehyde, ketone, protected ketone, thioester,ester, dicarbonyl, hydrazine, amidine, imine, diamine, keto-amine,keto-alkyne, or ene-dione. In certain embodiments of compounds ofFormula (I), V is a hydroxylamine, methyl, aldehyde, protected aldehyde,ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine,amidine, imine, diamine, keto-amine, keto-alkyne, and ene-dione.

In certain embodiments of compounds of Formula (I), each L isindependently a cleavable linker or non-cleavable linker. In certainembodiments of compounds of Formula (I), each L is independently aoligo(ethylene glycol) derivatized linker.

In certain embodiments of compounds of Formula (I), alkylene is —CH₂—,—CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH—,—CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, or—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—. In certain embodiments ofcompounds of Formula (I), each n, n′, n″, n′″, and n″″ is 0, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100.

In certain embodiments, dolastatin linker derivatives include compoundshaving the structure of Formula (II):

In some embodiments of compounds of Formula (II), L is-(alkylene-O)_(n)-alkylene-. In some embodiments, each alkylene is—CH₂CH₂—, n is equal to 3, and R₇ is methyl. In some embodiments, L is-alkylene-. In some embodiments of compounds of Formula (II), eachalkylene is —CH₂CH₂— and R₇ is methyl or hydrogen. In some embodimentsof compounds of Formula (II), L is -(alkylene-O)_(n)-alkylene-C(O)—. Insome embodiments of compounds of Formula (II), each alkylene is—CH₂CH₂—, n is equal to 4, and R₇ is methyl. In some embodiments ofcompounds of Formula (II), L is-(alkylene-O)_(n)—(CH₂)_(n′)—NHC(O)—(CH₂)_(n″)—C(Me)₂-S—S—(CH₂)_(n′″)—NHC(O)-(alkylene-O)_(n″″)-alkylene-.In some embodiments of compounds of Formula (II), each alkylene is—CH₂CH₂—, n is equal to 1, n′ is equal to 2, n″ is equal to 1, n′″ isequal to 2, n″″ is equal to 4, and R₇ is methyl. Such dolastatin linkerderivatives may be in the form of a salt, or may be incorporated into anon-natural amino acid polypeptide, polymer, polysaccharide, or apolynucleotide and optionally post translationally modified.

In certain embodiments of compounds of Formula (II), each L isindependently a cleavable linker or non-cleavable linker. In certainembodiments of compounds of Formula (II), each L is independently aoligo(ethylene glycol) derivatized linker.

In certain embodiments of compounds of Formula (II), R₇ is methyl,ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl,pentyl, or hexyl. In certain embodiments of compounds of Formula (II),R₇ is hydrogen.

In certain embodiments of compounds of Formula (II), alkylene is —CH₂—,—CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, orCH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—. In certain embodiments ofcompounds of Formula (II), each n, n′, n″, n′″, and n′″ is 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100.

Such dolastatin linker derivatives include compounds having thestructure of Formula (III), (IV), (V) or (VI):

wherein:

-   -   Z has the structure of:

-   -   -   R₅ is H, COR₈, C₁-C₆alkyl, or thiazole;            -   R₈ is OH;        -   R₆ is OH or H;        -   Ar is phenyl or pyridine;

    -   R₇ is C₁-C₆alkyl or hydrogen;

    -   Y is NH₂—O—;

    -   V is —O—NH₂

    -   L₁, L₂, L₃, and L₄ are each linkers independently selected from        the group consisting of a bond, -alkylene-,        -(alkylene-O)_(n)-alkylene-J-,        -alkylene′-J-(alkylene-O)_(n)-alkylene-,        -J-(alkylene-O)_(n)-alkylene-,        -(alkylene-O)_(n)-alkylene-J-(alkylene-O)_(n)′-alkylene-J′-,        -(alkylene-O)_(n)-alkylene-J-alkylene′-, —W—, -alkylene-W—,        alkylene′-J-(alkylene-NMe)_(n)-alkylene-W—,        -J-(alkylene-NMe)_(n)-alkylene-W—,        -J-alkylene-NMe-alkylene′-NMe-alkylene″-W—, and        -alkylene-J-alkylene′-NMe-alkylene″-NMe-alkylene′″-W—;        -   W has the structure of:

-   -   -   each J and J′ independently have the structure of:

-   -   -   each n and n′ are independently integers greater than or            equal to one.            Such dolastatin linker derivatives may be in the form of a            salt, or may be incorporated into a non-natural amino acid            polypeptide, polymer, polysaccharide, or a polynucleotide            and optionally post translationally modified.

In certain embodiments of compounds of Formula (III), (IV), (V) or (VI),R₅ is thiazole. In certain embodiments of compounds of Formula (III),(IV), (V) or (VI), R₆ is H. In certain embodiments of compounds ofFormula (III), (IV), (V) or (VI), Ar is phenyl. In certain embodimentsof compounds of Formula (III), (IV), (V) or (VI), R₇ is methyl. Incertain embodiments of compounds of Formula (III), (IV), (V) or (VI), nand n′ are integers from 0 to 20. In certain embodiments of compounds ofFormula (III), (IV), (V) or (VI), n and n′ are integers from 0 to 10. Incertain embodiments of compounds of Formula (III), (IV), (V) or (VI), nand n′ are integers from 0 to 5.

In certain embodiments of compounds of Formula (III) and (V), R₅ isthiazole or carboxylic acid. In certain embodiments of compounds ofFormula (III) and (V), R₅ is hydrogen. In certain embodiments ofcompounds of Formula (III) and (V), R₅ is methyl, ethyl, propyl,iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl.In certain embodiments of compounds of Formula (III) and (V), R₅ is—NH-(alkylene-O)_(n)—NH₂, wherein alkylene is —CH₂—, —CH₂CH₂—,—CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, or—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—. In certain embodiments ofcompounds to of Formula (III) and (V), R₅ is —NH-(alkylene-O)_(n)—NH₂,wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments of compounds of Formula (III), (IV), (V) and(VI), R₆ is H. In some embodiments of compounds of Formula (III), (IV),(V) and (VI), R₆ is hydroxy.

In certain embodiments of compounds of Formula (III), (IV), (V) and(VI), Ar is phenyl.

In certain embodiments of compounds of Formula (III), (IV), (V) and(VI), R₇ is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyliso-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments ofcompounds of Formula (III), (IV), (V) and (VI), R₇ is hydrogen.

In certain embodiments of compounds of Formula (III) and (V), Y ishydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone,thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine,keto-amine, keto-alkyne, or ene-dione. In certain embodiments ofcompounds of Formula (IV) and (VI), V is a hydroxylamine, methyl,aldehyde, protected aldehyde, ketone, protected ketone, thioester,ester, dicarbonyl, hydrazine, amidine, imine, diamine, keto-amine,keto-alkyne, and ene-dione.

In certain embodiments of compounds of Formula (XIV), (XV), (XVI),(XVII), and (XVIII), each L, L₁, L₂, L₃, and L₄ is independently acleavable linker or non-cleavable linker. In certain embodiments ofcompounds of Formula (XIV), (XV), (XVI), (XVII), and (XVIII), each L,L₁, L₂, L₃, and L₄ is independently a oligo(ethylene glycol) derivatizedlinker.

In certain embodiments of compounds of Formula (III), (IV), (V) and(VI), each alkylene, alkylene′, alkylene″, and alkylene′″ independentlyis —CH—, —CH₂CH₂—, —CH₂CH₂CH—, —CH₂CH₂CH₂CH₂, —CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, or—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—. In certain embodiments ofcompounds of Formula (III), (IV), (V) and (VI), alkylene is methylene,ethylene, propylene, butylenes, pentylene, hexylene, or heptylene.

In certain embodiments of compounds of Formula (III), (IV), (V) and(VI), each n and n′ independently is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100.

In certain embodiments, dolastatin linker derivatives include compoundshaving the structure of Formula (VII):

In certain embodiments of compounds of Formula (VII), L₁ is-(alkylene-O)_(n)-alkylene-J-, L₂ is-alkylene′-J′-(alkylene-O)_(n′)-alkylene-, L₃ is-J″-(alkylene-O)_(n)″-alkylene-, alkylene is —CH₂CH₂—, alkylene′ is—(CH₂)₄—, n is 1, n′ and n″ are 3, J has the structure of

J′ and J″ have the structure of

and R₇ is methyl. In certain embodiments of compounds of Formula (VII),L₁ is -J-(alkylene-O)_(n)-alkylene-, L₂ is-(alkylene-O)_(n′)-alkylene-J′-alkylene′-, L₃ is-(alkylene-O)_(n″)-alkylene-J″-, alkylene is —CH₂CH₂—, alkylene′ is—(CH₂)₄—, n is 1, n′ and n″ are 4, and J, J′ and J″ have the structureof

Such dolastatin linker derivatives may be in the form of a salt, or maybe incorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

In certain embodiments, compounds of Formula (I)-(VII) are stable inaqueous solution for at least 1 month under mildly acidic conditions. Incertain embodiments, compounds of Formula (I)-(VII) are stable for atleast 2 weeks under mildly acidic conditions. In certain embodiments,compound of Formula (I)-(VII) are stable for at least 5 days undermildly acidic conditions. In certain embodiments, such acidic conditionsare pH 2 to 8.

The methods and compositions provided and described herein includepolypeptides comprising dolastatin linker derivative containing at leastone carbonyl or dicarbonyl group, oxime group, hydroxylamine group, orprotected or masked forms thereof. Introduction of at least one reactivegroup into a dolastatin linker derivative can allow for the applicationof conjugation chemistries that involve specific chemical reactions,including, but not limited to, with one or more dolastatin linkerderivative(s) while not reacting with the commonly occurring aminoacids. Once incorporated, the dolastatin linker derivative side chainscan also be modified by utilizing chemistry methodologies describedherein or suitable for the particular functional groups or substituentspresent in the dolastatin linker derivative.

The dolastatin linker derivative methods and compositions describedherein provide conjugates of substances having a wide variety offunctional groups, substituents or moieties, with other substancesincluding but not limited to a polymer; a water-soluble polymer; aderivative of polyethylene glycol; a second protein or polypeptide orpolypeptide analog; an antibody or antibody fragment; and anycombination thereof.

In certain embodiments, the dolastatin linker derivatives, linkers andreagents described herein, including compounds of Formulas (I)-(VII) arestable in aqueous solution under mildly acidic conditions (including butnot limited to pH 2 to 8). In other embodiments, such compounds arestable for at least one month under mildly acidic conditions. In otherembodiments, such compounds are stable for at least 2 weeks under mildlyacidic conditions. In other embodiments, such compounds are stable forat least 5 days under mildly acidic conditions.

In another aspect of the compositions, methods, techniques andstrategies described herein are methods for studying or using any of theaforementioned “modified or unmodified” non-natural amino aciddolastatin linker derivatives. Included within this aspect, by way ofexample only, are therapeutic, diagnostic, assay-based, industrial,cosmetic, plant biology, environmental, energy-production,consumer-products, and/or military uses which would benefit from adolastatin linker derivative comprising a “modified or unmodified”non-natural amino acid polypeptide or protein.

Non-limiting examples of dolastatin linker derivatives include:

In another aspect of the present invention, the drug conjugated to theanti-CD70 antibody is dexamethasone. In another aspect of the presentinvention, the drug conjugated to the anti-CD70 antibody is dasantinib.In another aspect of the present invention, the drug conjugated to theanti-CD70 antibody is FK506. In another aspect of the present invention,the drug conjugated to the anti-CD70 antibody is a derivative ofdexamethasone. In another aspect of the present invention, the drugconjugated to the anti-CD70 antibody is a derivative of dasantinib. Inanother aspect of the present invention, the drug conjugated to theanti-CD70 antibody is a derivative of FK506.

In other aspects of the present invention, the linker-drug portion ofthe anti-CD70 antibody drug conjugate is chosen from one of thefollowing non-limiting examples:

IV. Non-Natural Amino Acid Derivatives

The non-natural amino acids used in the methods and compositionsdescribed herein have at least one of the following four properties: (1)at least one functional group on the sidechain of the non-natural aminoacid has at least one characteristics and/or activity and/or reactivityorthogonal to the chemical reactivity of the 20 common,genetically-encoded amino acids (i.e., alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine), or at least orthogonal tothe chemical reactivity of the naturally occurring amino acids presentin the polypeptide that includes the non-natural amino acid; (2) theintroduced non-natural amino acids are substantially chemically inerttoward the 20 common, genetically-encoded amino acids; (3) thenon-natural amino acid can be stably incorporated into a polypeptide,preferably with the stability commensurate with the naturally-occurringamino acids or under typical physiological conditions, and furtherpreferably such incorporation can occur via an in vivo system; and (4)the non-natural amino acid includes an oxime functional group or afunctional group that can be transformed into an oxime group by reactingwith a reagent, preferably under conditions that do not destroy thebiological properties of the polypeptide that includes the non-naturalamino acid (unless of course such a destruction of biological propertiesis the purpose of the modification/transformation), or where thetransformation can occur under aqueous conditions at a pH between about4 and about 8, or where the reactive site on the non-natural amino acidis an electrophilic site. Any number of non-natural amino acids can beintroduced into the polypeptide. Non-natural amino acids may alsoinclude protected or masked oximes or protected or masked groups thatcan be transformed into an oxime group after deprotection of theprotected group or unmasking of the masked group. Non-natural aminoacids may also include protected or masked carbonyl or dicarbonylgroups, which can be transformed into a carbonyl or dicarbonyl groupafter deprotection of the protected group or unmasking of the maskedgroup and thereby are available to react with hydroxylamines or oximesto form oxime groups.

Non-natural amino acids that may be used in the methods and compositionsdescribed herein include, but are not limited to, amino acids comprisinga amino acids with novel functional groups, amino acids that covalentlyor noncovalently interact with other molecules, glycosylated amino acidssuch as a sugar substituted serine, other carbohydrate modified aminoacids, keto-containing amino acids, aldehyde-containing amino acids,amino acids comprising polyethylene glycol or other polyethers, heavyatom substituted amino acids, chemically cleavable and/or photocleavableamino acids, amino acids with an elongated side chains as compared tonatural amino acids, including but not limited to, polyethers or longchain hydrocarbons, including but not limited to, greater than about 5or greater than about 10 carbons, carbon-linked sugar-containing aminoacids, redox-active amino acids, amino thioacid containing amino acids,and amino acids comprising one or more toxic moiety.

In some embodiments, non-natural amino acids comprise a saccharidemoiety. Examples of such amino acids includeN-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,N-acetyl-L-glucosaminyl-L-threonine,N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.Examples of such amino acids also include examples where thenaturally-occurring N- or O-linkage between the amino acid and thesaccharide is replaced by a covalent linkage not commonly found innature—including but not limited to, an alkene, an oxime, a thioether,an amide and the like. Examples of such amino acids also includesaccharides that are not commonly found in naturally-occurring proteinssuch as 2-deoxy-glucose, 2-deoxygalactose and the like.

The chemical moieties incorporated into polypeptides via incorporationof non-natural amino acids into such polypeptides offer a variety ofadvantages and manipulations of polypeptides. For example, the uniquereactivity of a carbonyl or dicarbonyl functional group (including aketo- or aldehyde-functional group) allows selective modification ofproteins with any of a number of hydrazine- or hydroxylamine-containingreagents in vivo and in vitro. A heavy atom non-natural amino acid, forexample, can be useful for phasing x-ray structure data. Thesite-specific introduction of heavy atoms using non-natural amino acidsalso provides selectivity and flexibility in choosing positions forheavy atoms. Photoreactive non-natural amino acids (including but notlimited to, amino acids with benzophenone and arylazides (including butnot limited to, phenylazide) side chains), for example, allow forefficient in vivo and in vitro photocrosslinking of polypeptides.Examples of photoreactive non-natural amino acids include, but are notlimited to, p-azido-phenylalanine and p-benzoyl-phenylalanine. Thepolypeptide with the photoreactive non-natural amino acids may then becrosslinked at will by excitation of the photoreactive group-providingtemporal control. In a non-limiting example, the methyl group of anon-natural amino can be substituted with an isotopically labeled,including but not limited to, with a methyl group, as a probe of localstructure and dynamics, including but not limited to, with the use ofnuclear magnetic resonance and vibrational spectroscopy.

A. Structure and Synthesis of Non-Natural Amino Acid Derivatives:Carbonyl, Carbonyl like, Masked Carbonyl, and Protected Carbonyl Groups

Amino acids with an electrophilic reactive group allow for a variety ofreactions to link molecules via various chemical reactions, including,but not limited to, nucleophilic addition reactions. Such electrophilicreactive groups include a carbonyl- or dicarbonyl-group (including aketo- or aldehyde group), a carbonyl-like- or dicarbonyl-like-group(which has reactivity similar to a carbonyl- or dicarbonyl-group and isstructurally similar to a carbonyl- or dicarbonyl-group), a maskedcarbonyl- or masked dicarbonyl-group (which can be readily convertedinto a carbonyl- or dicarbonyl-group), or a protected carbonyl- orprotected dicarbonyl-group (which has reactivity similar to a carbonyl-or dicarbonyl-group upon deprotection). Such amino acids include aminoacids having the structure of Formula (XXXVII):

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   K is

-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   each R″ is independently H, alkyl, substituted alkyl, or a    protecting group, or when more than one R″ group is present, two R″    optionally form a heterocycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   each of R₃ and R₄ is independently H, halogen, lower alkyl, or    substituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally    form a cycloalkyl or a heterocycloalkyl;-   or the -A-B—K—R groups together form a bicyclic or tricyclic    cycloalkyl or heterocycloalkyl comprising at least one carbonyl    group, including a dicarbonyl group, protected carbonyl group,    including a protected dicarbonyl group, or masked carbonyl group,    including a masked dicarbonyl group;-   or the —K—R group together forms a monocyclic or bicyclic cycloalkyl    or heterocycloalkyl comprising at least one carbonyl group,    including a dicarbonyl group, protected carbonyl group, including a    protected dicarbonyl group, or masked carbonyl group, including a    masked dicarbonyl group;    with a proviso that when A is phenylene and each R₃ is H, B is    present; and that when A is —(CH₂)₄— and each R₃ is H, B is not    —NHC(O)(CH₂CH₂)—; and that when A and B are absent and each R₃ is H,    R is not methyl. Such non-natural amino acids may be in the form of    a salt, or may be incorporated into a non-natural amino acid    polypeptide, polymer, polysaccharide, or a polynucleotide and    optionally post translationally modified.

In certain embodiments, compounds of Formula (XXXVII) are stable inaqueous solution for at least 1 month under mildly acidic conditions. Incertain embodiments, compounds of Formula (XXXVII) are stable for atleast 2 weeks under mildly acidic conditions. In certain embodiments,compound of Formula (XXXVII) are stable for at least 5 days under mildlyacidic conditions. In certain embodiments, such acidic conditions are pH2 to 8.

In certain embodiments of compounds of Formula (XXXVII), B is loweralkylene, substituted lower alkylene, —O-(alkylene or substitutedalkylene)-, —C(R′)═N—N(R′)—, —N(R′)CO—, —C(O)—, —C(R′)═N—,—C(O)-(alkylene or substituted alkylene)-, —CON(R′)-(alkylene orsubstituted alkylene)-, —S(alkylene or substituted alkylene)-,—S(O)(alkylene or substituted alkylene)-, or —S(O)₂(alkylene orsubstituted alkylene)-. In certain embodiments of compounds of Formula(XXXVII), B is —O(CH₂)—, —CH═N—, —CH═N—NH—, —NHCH₂—, —NHCO—, —C(O)—,—C(O)—(CH₂)—, —CONH—(CH₂)—, —SCH₂—, —S(═O)CH₂—, or —S(O)₂CH₂—. Incertain embodiments of compounds of Formula (XXXVII), R is C₁₋₆ alkyl orcycloalkyl. In certain embodiments of compounds of Formula (XXXVII) R is—CH₃, —CH(CH₃)₂, or cyclopropyl. In certain embodiments of compounds ofFormula (XXXVII), R₁ is H, tert-butyloxycarbonyl (Boc),9-Fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA), orbenzyloxycarbonyl (Cbz). In certain embodiments of compounds of Formula(XXXVII), R₁ is a resin, amino acid, polypeptide, antibody, orpolynucleotide. In certain embodiments of compounds of Formula (XXXVII),R₂ is OH, O-methyl, O-ethyl, or O-t-butyl. In certain embodiments ofcompounds of Formula (XXXVII), R₂ is a resin, amino acid, polypeptide,antibody, or polynucleotide. In certain embodiments of compounds ofFormula (XXXVII), R₂ is a polynucleotide. In certain embodiments ofcompounds of Formula (XXXVII), R₂ is ribonucleic acid (RNA).

In certain embodiments of compounds of Formula (XXXVII),

is selected from the group consisting of:

-   -   (i) A is substituted lower alkylene, C₄-arylene, substituted        arylene, heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;        -   B is optional, and when present is a divalent linker            selected from the group consisting of lower alkylene,            substituted lower alkylene, lower alkenylene, substituted            lower alkenylene, —O—, —O-(alkylene or substituted            alkylene)-, —S—, —S(O)—, —S(O)₂—, —NS(O)₂—, —OS(O)₂—,            —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,            —N(R′)—, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted            alkylene)-, —CSN(R′)—, —N(R′)CO-(alkylene or substituted            alkylene)-, —N(R′)C(O)O—, —N(R′)C(S)—, —S(O)N(R′),            —S(O)₂N(R′), —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,            —N(R′)S(O)N(R′)—, —N(R′)S(O)₂N(R′)—, —N(R′)—N═,            —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and            —C(R′)₂—N(R′)—N(R′)—;    -   (ii) A is optional, and when present is substituted lower        alkylene, C₄-arylene, substituted arylene, heteroarylene,        substituted heteroarylene, alkarylene, substituted alkarylene,        aralkylene, or substituted aralkylene;        -   B is a divalent linker selected from the group consisting of            lower alkylene, substituted lower alkylene, lower            alkenylene, substituted lower alkenylene, —O—, —O-(alkylene            or substituted alkylene)-, —S—, —S(O)—, —S(O)₂—, —NS(O)₂—,            —OS(O)₂—, —C(O)—, —C(O)-(alkylene or substituted alkylene)-,            —C(S)—, —N(R′)—, —C(O)N(R′)—, —CON(R′)-(alkylene or            substituted alkylene)-, —CSN(R′)—, —N(R′)CO-(alkylene or            substituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(S)—,            —S(O)N(R′), —S(O)₂N(R′), —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,            —N(R′)S(O)N(R′)—, —N(R′)S(O)₂N(R′)—, —N(R′)—N═,            —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and            —C(R′)₂—N(R′)—N(R′)—;    -   (iii) A is lower alkylene;        -   B is optional, and when present is a divalent linker            selected from the group consisting of lower alkylene,            substituted lower alkylene, lower alkenylene, substituted            lower alkenylene, —O—, —O-(alkylene or substituted            alkylene)-, —S—, —S(O)—, —S(O)₂—, —NS(O)₂—, —OS(O)₂—,            —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,            —N(R′)—, —C(O)N(R′)—, —CSN(R′)—, —CON(R′)-(alkylene or            substituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(S)—,            —S(O)N(R′), —S(O)₂N(R′), —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,            —N(R′)S(O)N(R′)—, —N(R′)S(O)₂N(R′)—, —N(R′)—N═,            —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and            —C(R′)₂—N(R′)—N(R′)—; and    -   (iv) A is phenylene;        -   B is a divalent linker selected from the group consisting of            lower alkylene, substituted lower alkylene, lower            alkenylene, substituted lower alkenylene, —O—, —O-(alkylene            or substituted alkylene)-, —S—, —S(O)—, —S(O)₂—, —NS(O)₂—,            —OS(O)₂—, —C(O)—, —C(O)-(alkylene or substituted alkylene)-,            —C(S)—, —N(R′)—, —C(O)N(R′)—, —CON(R′)-(alkylene or            substituted alkylene)-, —CSN(R′)—, —N(R′)CO-(alkylene or            substituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(S)—,            —S(O)N(R′), —S(O)₂N(R′), —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,            —N(R′)S(O)N(R′)—, —N(R′)S(O)₂N(R′)—, —N(R′)—N═,            —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and            —C(R′)₂—N(R′)—N(R′)—;    -   K is

-   -   each R′ is independently H, alkyl, or substituted alkyl;    -   R₁ is optional, and when present, is H, an amino protecting        group, resin, amino acid, polypeptide, or polynucleotide; and    -   R₂ is optional, and when present, is OH, an ester protecting        group, resin, amino acid, polypeptide, or polynucleotide; and    -   each R₃ and R₄ is independently H, halogen, lower alkyl, or        substituted lower alkyl;    -   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted        cycloalkyl;

In addition, amino acids having the structure of Formula (XXXVIII) areincluded:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, at least one amino acid,    polypeptide, or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, at least one amino acid,    polypeptide, or polynucleotide;    with a proviso that when A is phenylene, B is present; and that when    A is —(CH₂)₄—, B is not —NHC(O)(CH₂CH₂)—; and that when A and B are    absent, R is not methyl. Such non-natural amino acids may be in the    form of a salt, or may be incorporated into a non-natural amino acid    polypeptide, polymer, polysaccharide, or a polynucleotide and    optionally post translationally modified.

In addition, amino acids having the structure of Formula (XXXIX) areincluded:

wherein:

-   B is a linker selected from the group consisting of lower alkylene,    substituted lower alkylene, lower alkenylene, substituted lower    alkenylene, lower heteroalkylene, substituted lower heteroalkylene,    —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or    substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or 3,    —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—, —NS(O)₂—,    —OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,    —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene    or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or    substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted    alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-,    —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,    —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,    —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′    is independently H, alkyl, or substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;

each R_(a) is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2,or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is independentlyH, alkyl, or substituted alkyl. Such non-natural amino acids may be inthe form of a salt, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition, the following amino acids are included:

Such non-natural amino acids may be are optionally amino protectedgroup, carboxyl protected and/or in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

In addition, the following amino acids having the structure of Formula(XXXX) are included:

wherein

-   —NS(O)₂—, —OS(O)₂—, optional, and when present is a linker selected    from the group consisting of lower alkylene, substituted lower    alkylene, lower alkenylene, substituted lower alkenylene, lower    heteroalkylene, substituted lower heteroalkylene, —O—, —O-(alkylene    or substituted alkylene)-, —S—, —S-(alkylene or substituted    alkylene)-, —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene or    substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   each R_(a) is independently selected from the group consisting of H,    halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is    1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is    independently H, alkyl, or substituted alkyl; and n is 0 to 8;    with a proviso that when A is —(CH₂)₄—, B is not —NHC(O)(CH₂CH₂)—.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition, the following amino acids having the structure of Formula(XXXXI) are included:

wherein,

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids having the structure of Formula(XXXXII) are included:

wherein,

-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)—N═N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide; wherein each R_(a) is independently selected from    the group consisting of H, halogen, alkyl, substituted alkyl,    —N(R′)₂, —C(O)_(k)R′ where k is 1, 2, or 3, —C(O)N(R′)₂, —OR′, and    —S(O)_(k)R′, where each R′ is independently H, alkyl, or substituted    alkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition, the following amino acids having the structure of Formula(XXXXIV) are included:

wherein,

-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)N(R′)—, —N(R′)C(O)N(R′)—,    —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—,    —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   each R_(a) is independently selected from the group consisting of H,    halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is    1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is    independently H, alkyl, or substituted alkyl; and n is 0 to 8.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition to monocarbonyl structures, the non-natural amino acidsdescribed herein may include groups such as dicarbonyl, dicarbonyl like,masked dicarbonyl and protected dicarbonyl groups.

For example, the following amino acids having the structure of Formula(XXXXV) are included:

wherein,

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids having the structure of Formula(XXXXVI) are included:

wherein,

-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide; wherein each R_(a) is independently selected from    the group consisting of H, halogen, alkyl, substituted alkyl,    —N(R′)₂, —C(O)_(k)R′ where k is 1, 2, or 3, —C(O)N(R′)₂, —OR′, and    —S(O)_(k)R′, where each R′ is independently H, alkyl, or substituted    alkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected and carboxylprotected, or a salt thereof. Such non-natural amino acids may be in theform of a salt, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition, the following amino acids having the structure of Formula(XXXXVII) are included:

wherein,

-   B is optional, and when present is a linker selected from the group    consisting of lower alkylene, substituted lower alkylene, lower    alkenylene, substituted lower alkenylene, lower heteroalkylene,    substituted lower heteroalkylene, —O—, —O-(alkylene or substituted    alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—    where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,    —C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted    alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-,    —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,    —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,    —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or    substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,    —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,    —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and    —C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or    substituted alkyl;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide; each R^(a) is independently selected from the    group consisting of H, halogen, alkyl, substituted alkyl, —N(R′)₂,    —C(O)_(k)R′ where k is 1, 2, or 3, —C(O)N(R′)₂, —OR′, and    —S(O)_(k)R′, where each R′ is independently H, alkyl, or substituted    alkyl; and n is 0 to 8.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected and carboxylprotected, or a salt thereof, or may be incorporated into a non-naturalamino acid polypeptide, polymer, polysaccharide, or a polynucleotide andoptionally post translationally modified.

In addition, the following amino acids having the structure of Formula(XXXXVIII) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   X₁ is C, S, or S(O); and L is alkylene, substituted alkylene,    N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H,    alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids having the structure of Formula(XXXXIX) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   L is alkylene, substituted alkylene, N(R′)alkylene) or    N(R′)(substituted alkylene), where R′ is H, alkyl, substituted    alkyl, cycloalkyl, or substituted cycloalkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids having the structure of Formula(XXXXX) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   L is alkylene, substituted alkylene, N(R′)(alkylene) or    N(R′)(substituted alkylene), where R′ is H, alkyl, substituted    alkyl, cycloalkyl, or substituted cycloalkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids having the structure of Formula(XXXXXI) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   X₁ is C, S, or S(O); and n is 0, 1, 2, 3, 4, or 5; and each R⁸ and    R⁹ on each CR⁸R⁹ group is independently selected from the group    consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R⁸    and R⁹ can together form ═O or a cycloalkyl, or any to adjacent R⁸    groups can together form a cycloalkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids having the structure of Formula(XXXXXII) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide; n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ on    each CR⁸R⁹ group is independently selected from the group consisting    of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ can    together form ═O or a cycloalkyl, or any to adjacent R⁸ groups can    together form a cycloalkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids having the structure of Formula(XXXXXIII) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide; n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ on    each CR⁸R⁹ group is independently selected from the group consisting    of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ can    together form ═O or a cycloalkyl, or any to adjacent R⁸ groups can    together form a cycloalkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids having the structure of Formula(XXXXXIV) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   X₁ is C, S, or S(O); and L is alkylene, substituted alkylene,    N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H,    alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids having the structure of Formula(XXXXXV) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   L is alkylene, substituted alkylene, N(R′)(alkylene) or    N(R′)(substituted alkylene), where R′ is H, alkyl, substituted    alkyl, cycloalkyl, or substituted cycloalkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, the following amino acids having the structure of Formula(XXXXXVI) are included:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted    cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   L is alkylene, substituted alkylene, N(R′)(alkylene) or    N(R′)(substituted alkylene), where R′ is H, alkyl, substituted    alkyl, cycloalkyl, or substituted cycloalkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, amino acids having the structure of Formula (XXXXXVII) areincluded:

wherein:

-   A is optional, and when present is lower alkylene, substituted lower    alkylene, lower cycloalkylene, substituted lower cycloalkylene,    lower alkenylene, substituted lower alkenylene, alkynylene, lower    heteroalkylene, substituted heteroalkylene, lower    heterocycloalkylene, substituted lower heterocycloalkylene, arylene,    substituted arylene, heteroarylene, substituted heteroarylene,    alkarylene, substituted alkarylene, aralkylene, or substituted    aralkylene;-   M is —C(R₃)—,

where (a) indicates bonding to the A group and (b) indicates bonding torespective carbonyl groups, R₃ and R₄ are independently chosen from H,halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl, or R₃ and R₄ or two R₃ groups or two R₄ groups optionallyform a cycloalkyl or a heterocycloalkyl;

-   R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or    substituted cycloalkyl;-   T₃ is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl,    substituted alkyl, cycloalkyl, or substituted cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, amino acids having the structure of Formula (XXXXXVIII) areincluded:

wherein:

-   M is —C(R₃)—,

where (a) indicates bonding to the A group and (b) indicates bonding torespective carbonyl groups, R₃ and R₄ are independently chosen from H,halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl, or R₃ and R₄ or two R₃ groups or two R₄ groups optionallyform a cycloalkyl or a heterocycloalkyl;

-   R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or    substituted cycloalkyl;-   T₃ is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl,    substituted alkyl, cycloalkyl, or substituted cycloalkyl;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide; each R_(a) is independently selected from the    group consisting of H, halogen, alkyl, substituted alkyl, —N(R′)₂,    —C(O)_(k)R′ where k is 1, 2, or 3, —C(O)N(R′)₂, —OR′, and    —S(O)_(k)R′, where each R′ is independently H, alkyl, or substituted    alkyl.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, amino acids having the structure of Formula (XXXXXIX) areincluded:

wherein:

-   R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or    substituted cycloalkyl; and-   T₃ is O, or S.    Such non-natural amino acids may be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide and optionally post    translationally modified.

In addition, amino acids having the structure of Formula (XXXXXX) areincluded:

wherein:

-   R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or    substituted cycloalkyl.

In addition, the following amino acids having structures of Formula(XXXXXX) are included:

Such non-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

The carbonyl or dicarbonyl functionality can be reacted selectively witha hydroxylamine-containing reagent under mild conditions in aqueoussolution to form the corresponding oxime linkage that is stable underphysiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc.81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc.117(14):3893-3899 (1995). Moreover, the unique reactivity of thecarbonyl or dicarbonyl group allows for selective modification in thepresence of the other amino acid side chains. See, e.g., Cornish, V. W.,et al., J. Am. Chem. Soc. 118:8150-8151 (1996); Geoghegan, K. F. &Stroh, J. G., Bioconjug. Chem. 3:138-146 (1992); Mahal, L. K., et al.,Science 276:1125-1128 (1997).

The synthesis of p-acetyl-(+/−)-phenylalanine andm-acetyl-(+/−)-phenylalanine is described in Zhang, Z., et al.,Biochemistry 42: 6735-6746 (2003), incorporated by reference. Othercarbonyl- or dicarbonyl-containing amino acids can be similarlyprepared.

In some embodiments, a polypeptide comprising a non-natural amino acidis chemically modified to generate a reactive carbonyl or dicarbonylfunctional group. For instance, an aldehyde functionality useful forconjugation reactions can be generated from a functionality havingadjacent amino and hydroxyl groups. Where the biologically activemolecule is a polypeptide, for example, an N-terminal serine orthreonine (which may be normally present or may be exposed via chemicalor enzymatic digestion) can be used to generate an aldehydefunctionality under mild oxidative cleavage conditions using periodate.See, e.g., Gaertner, et. al., Bioconjug. Chem. 3: 262-268 (1992);Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertneret al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known inthe art are restricted to the amino acid at the N-terminus of thepeptide or protein.

Additionally, by way of example a non-natural amino acid bearingadjacent hydroxyl and amino groups can be incorporated into apolypeptide as a “masked” aldehyde functionality. For example,5-hydroxylysine bears a hydroxyl group adjacent to the epsilon amine.Reaction conditions for generating the aldehyde typically involveaddition of molar excess of sodium metaperiodate under mild conditionsto avoid oxidation at other sites within the polypeptide. The pH of theoxidation reaction is typically about 7.0. A typical reaction involvesthe addition of about 1.5 molar excess of sodium meta periodate to abuffered solution of the polypeptide, followed by incubation for about10 minutes in the dark. See, e.g. U.S. Pat. No. 6,423,685.

B. Structure and Synthesis of Non-Natural Amino Acids: Dicarbonyl,Dicarbonyl-Like, Masked Dicarbonyl, and Protected Dicarbonyl Groups

Amino acids with an electrophilic reactive group allow for a variety ofreactions to link molecules via nucleophilic addition reactions amongothers. Such electrophilic reactive groups include a dicarbonyl group(including a diketone group, a ketoaldehyde group, a ketoacid group, aketoester group, and a ketothioester group), a dicarbonyl-like group(which has reactivity similar to a dicarbonyl group and is structurallysimilar to a dicarbonyl group), a masked dicarbonyl group (which can bereadily converted into a dicarbonyl group), or a protected dicarbonylgroup (which has reactivity similar to a dicarbonyl group upondeprotection). Such amino acids include amino acids having the structureof Formula (XXXVII):

wherein:

-   -   A is optional, and when present is lower alkylene, substituted        lower alkylene, lower cycloalkylene, substituted lower        cycloalkylene, lower alkenylene, substituted lower alkenylene,        alkynylene, lower heteroalkylene, substituted heteroalkylene,        lower heterocycloalkylene, substituted lower        heterocycloalkylene, arylene, substituted arylene,        heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;    -   B is optional, and when present is a linker linked at one end to        a diamine containing moiety, the linker selected from the group        consisting of lower alkylene, substituted lower alkylene, lower        alkenylene, substituted lower alkenylene, lower heteroalkylene,        substituted lower heteroalkylene, —O-(alkylene or substituted        alkylene)-, —S-(alkylene or substituted alkylene)-, —C(O)R″—,        —S(O)_(k)(alkylene or substituted alkylene)-, where k is 1, 2,        or 3, —C(O)-(alkylene or substituted alkylene)-, —C(S)-(alkylene        or substituted alkylene)-, —NR″-(alkylene or substituted        alkylene)-, —CON(R″)-(alkylene or substituted alkylene)-,        —CSN(R″)-(alkylene or substituted alkylene)-, and        —N(R″)CO-(alkylene or substituted alkylene)-, where each R″ is        independently H, alkyl, or substituted alkyl;    -   K is

where,

-   -   T₁ is a bond, optionally substituted C₁-C₄ alkylene, optionally        substituted C₁-C₄ alkenylene, or optionally substituted        heteroalkyl;    -   wherein each optional substituents is independently selected        from lower alkylene, substituted lower alkylene, lower        cycloalkylene, substituted lower cycloalkylene, lower        alkenylene, substituted lower alkenylene, alkynylene, lower        heteroalkylene, substituted heteroalkylene, lower        heterocycloalkylene, substituted lower heterocycloalkylene,        arylene, substituted arylene, heteroarylene, substituted        heteroarylene, alkarylene, substituted alkarylene, aralkylene,        or substituted aralkylene;    -   T₂, is selected from the group consisting of lower alkylene,        substituted lower alkylene, lower alkenylene, substituted lower        alkenylene, lower heteroalkylene, substituted lower        heteroalkylene, —O—, —O-(alkylene or substituted alkylene)-,        —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k        is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,        —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,        —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,        —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,        —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,        —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene        or substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,        —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—,        —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═,        —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ is        independently H, alkyl, or substituted alkyl;    -   T₃ is

where each X₁ is independently selected from the group consisting of—O—, —S—, —N(H)—, —N(R)—, —N(Ac)—, and —N(OMe)-; X₂ is —OR, —OAc, —SR,—N(R)₂, —N(R)(Ac), —N(R)(OMe), or N₃, and where each R′ is independentlyH, alkyl, or substituted alkyl;

-   -   R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or        substituted cycloalkyl;    -   R₁ is H, an amino protecting group, resin, amino acid,        polypeptide, or polynucleotide; and    -   R₂ is OH, an ester protecting group, resin, amino acid,        polypeptide, or polynucleotide;    -   or the -A-B—K—R groups together form a bicyclic or tricyclic        cycloalkyl or heterocycloalkyl comprising at least one carbonyl        group, including a dicarbonyl group, protected carbonyl group,        including a protected dicarbonyl group, or masked carbonyl        group, including a masked dicarbonyl group;    -   or the —K—R group together forms a monocyclic or bicyclic        cycloalkyl or heterocycloalkyl comprising at least one carbonyl        group, including a dicarbonyl group, protected carbonyl group,        including a protected dicarbonyl group, or masked carbonyl        group, including a masked dicarbonyl group.

Non-limiting example of dicarbonyl amino acids having the structure ofFormula (XXXVII) include:

The following amino acids having structures of Formula (XXXVII) are alsoincluded:

Such non-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

C. Structure and Synthesis of Non-Natural Amino Acids: Ketoalkyne,Ketoalkyne-Like, Masked Ketoalkyne, Protected Ketoalkyne Group, Alkyne,and Cycloalkyne Groups

Amino acids containing reactive groups with dicarbonyl-like reactivityallow for the linking of molecules via nucleophilic addition reactions.Such electrophilic reactive reactiveonally groups include a ketoalkynegroup, a ketoalkyne-like group (which has reactivity similar to aketoalkyne group and is structurally similar to a ketoalkyne group), amasked ketoalkyne group (which can be readily converted into aketoalkyne group), or a protected ketoalkyne group (which has reactivitysimilar to a ketoalkyne group upon deprotection). In some embodiments,amino acids containing reactive groups with a terminal alkyne, internalalkyne or cycloalkyne allow for linking of molecules via cycloadditionreactions (e.g., 1,3-dipolar cycloadditions, azide-alkyne Huisgencycloaddition, etc.) Such amino acids include amino acids having thestructure of Formula (XXXXXXI-A) or (XXXXXXI-B):

wherein:

-   -   A is optional, and when present is lower alkylene, substituted        lower alkylene, lower cycloalkylene, substituted lower        cycloalkylene, lower alkenylene, substituted lower alkenylene,        alkynylene, lower heteroalkylene, substituted heteroalkylene,        lower heterocycloalkylene, substituted lower        heterocycloalkylene, arylene, substituted arylene,        heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;    -   B is optional, and when present is a linker linked at one end to        a diamine containing moiety, the linker selected from the group        consisting of lower alkylene, substituted lower alkylene, lower        alkenylene, substituted lower alkenylene, lower heteroalkylene,        substituted lower heteroalkylene, —O-(alkylene or substituted        alkylene)-, —S-(alkylene or substituted alkylene)-, —C(O)R″—,        —S(O)_(k)(alkylene or substituted alkylene)-, where k is 1, 2,        or 3, —C(O)-(alkylene or substituted alkylene)-, —C(S)-(alkylene        or substituted alkylene)-, —NR″-(alkylene or substituted        alkylene)-, —CON(R″)-(alkylene or substituted alkylene)-,        —CSN(R″)-(alkylene or substituted alkylene)-, and        —N(R″)CO-(alkylene or substituted alkylene)-, where each R″ is        independently H, alkyl, or substituted alkyl;    -   G is optional, and when present is

-   -   T₄ is a carbonyl protecting group including, but not limited to

where each X₁ is independently selected from the group consisting of—O—, —S—, —N(H)—, —N(R)—, —N(Ac)—, and —N(OMe)-; X₂ is —OR, —OAc, —SR,—N(R)₂, —N(R)(Ac), —N(R)(OMe), or N₃, and where each R′ is independentlyH, alkyl, or substituted alkyl;

-   -   R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or        substituted cycloalkyl;    -   R₁ is H, an amino protecting group, resin, amino acid,        polypeptide, or polynucleotide;    -   R₂ is OH, an ester protecting group, resin, amino acid,        polypeptide, or polynucleotide;    -   each of R₃ and R₄ is independently H, halogen, lower alkyl, or        substituted lower alkyl, or R₃ and R₄ or two R₃ groups        optionally form a cycloalkyl or a heterocycloalkyl;    -   each R₁₉ is independently selected from the group consisting of        C₁-C₆ alkyl, C₁-C₆ alkoxy, ester, ether, thioether, aminoalkyl,        halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl        halide, alkyl amine, alkyl sulfonic acid, alkyl nitro,        thioester, sulfonyl ester, halosulfonyl, nitrile, alkyl nitrile,        and nitro; and    -   q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.

D. Structure and Synthesis of Non-Natural Amino Acids: Ketoamine,Ketoamine-like, Masked Ketoamine, and Protected Ketoamine Groups

Amino acids containing reactive groups with dicarbonyl-like reactivityallow for the linking of molecules via nucleophilic addition reactions.Such reactive groups include a ketoamine group, a ketoamine-like group(which has reactivity similar to a ketoamine group and is structurallysimilar to a ketoamine group), a masked ketoamine group (which can bereadily converted into a ketoamine group), or a protected ketoaminegroup (which has reactivity similar to a ketoamine group upondeprotection). Such amino acids include amino acids having the structureof Formula (XXXXXXII):

wherein:

-   -   A is optional, and when present is lower alkylene, substituted        lower alkylene, lower cycloalkylene, substituted lower        cycloalkylene, lower alkenylene, substituted lower alkenylene,        alkynylene, lower heteroalkylene, substituted heteroalkylene,        lower heterocycloalkylene, substituted lower        heterocycloalkylene, arylene, substituted arylene,        heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;    -   B is optional, and when present is a linker linked at one end to        a diamine containing moiety, the linker selected from the group        consisting of lower alkylene, substituted lower alkylene, lower        alkenylene, substituted lower alkenylene, lower heteroalkylene,        substituted lower heteroalkylene, —O-(alkylene or substituted        alkylene)-, —S-(alkylene or substituted alkylene)-, —C(O)R″—,        —S(O)_(k)(alkylene or substituted alkylene)-, where k is 1, 2,        or 3, —C(O)-(alkylene or substituted alkylene)-, —C(S)-(alkylene        or substituted alkylene)-, —NR″-(alkylene or substituted        alkylene)-, —CON(R″)-(alkylene or substituted alkylene)-,        —CSN(R″)-(alkylene or substituted alkylene)-, and        —N(R″)CO-(alkylene or substituted alkylene)-, where each R″ is        independently H, alkyl, or substituted alkyl;    -   G is

-   -   T₁ is an optionally substituted C₁-C₄ alkylene, an optionally        substituted C₁-C₄ alkenylene, or an optionally substituted        heteroalkyl;    -   T₄ is a carbonyl protecting group including, but not limited to,

where each X₁ is independently selected from the group consisting of—O—, —S—, —N(H)—, —N(R′)—, —N(Ac)—, and —N(OMe)-; X₂ is —OR, —OAc, —SR,—N(R′)₂, —N(R′)(Ac), —N(R′)(OMe), or N₃, and where each R′ isindependently H, alkyl, or substituted alkyl;

-   -   R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or        substituted cycloalkyl;    -   R₁ is H, an amino protecting group, resin, amino acid,        polypeptide, or polynucleotide; and    -   R₂ is OH, an ester proiecting group, resin, amino acid,        polypeptide, or polynucleotide;    -   each of R₃ and R₄ is independently H, halogen, lower alkyl, or        substituted lower alkyl, or R₃ and R₄ or two R₃ groups        optionally form a cycloalkyl or a heterocycloalkyl.

Amino acids having the structure of Formula (XXXXXXII) include aminoacids having the structure of Formula (XXXXXXII) and Formula (XXXXXXIV):

-   -   wherein each R_(a) is independently selected from the group        consisting of H, halogen, alkyl, substituted alkyl, —N(R′)₂,        —C(O)_(k)R′ where k is 1, 2, or 3, —C(O)N(R′)₂, —OR′, and        —S(O)_(k)R′, where each R′ is independently H, alkyl, or        substituted alkyl.

E. Structure and Synthesis of Non-Natural Amino Acids: Diamine,Diamine-like, Masked Diamine, Protected Amines and Azides

Amino acids with a nucleophilic reactive group allow for a variety ofreactions to link molecules via electrophilic addition reactions amongothers. Such nucleophilic reactive groups include a diamine group(including a hydrazine group, an amidine group, an imine group, a1,1-diamine group, a 1,2-diamine group, a 1,3-diamine group, and a1,4-diamine group), a diamine-like group (which has reactivity similarto a diamine group and is structurally similar to a diamine group), amasked diamine group (which can be readily converted into a diaminegroup), or a protected diamine group (which has reactivity similar to adiamine group upon deprotection). In some embodiments, amino acidscontaining reactive groups with azides allow for linking of moleculesvia cycloaddition reactions (e.g., 1,3-dipolar cycloadditions,azide-alkyne Huisgen cycloaddition, etc.).

In another aspect are methods for the chemical synthesis ofhydrazine-substituted molecules for the derivatization ofcarbonyl-substituted dolastatin derivatives. In one embodiment, thehydrazine-substituted molecule can dolastatin linked derivatives. In oneembodiment are methods for the preparation of hydrazine-substitutedmolecules suitable for the derivatization of carbonyl-containingnon-natural amino acid polypeptides, including by way of example only,ketone-, or aldehyde-containing non-natural amino acid polypeptides. Ina further or additional embodiment, the non-natural amino acids areincorporated site-specifically during the in vivo translation ofproteins. In a further or additional embodiment, thehydrazine-substituted dolastatin derivatives allow for the site-specificderivatization of carbonyl-containing non-natural amino acids vianucleophilic attack of each carbonyl group to form aheterocycle-derivatized polypeptide, including a nitrogen-containingheterocycle-derivatized polypeptide in a site-specific fashion. In afurther or additional embodiment, the method for the preparation ofhydrazine-substituted dolastatin derivatives provides access to a widevariety of site-specifically derivatized polypeptides. In a further oradditional embodiment are methods for synthesizinghydrazine-functionalized polyethyleneglycol (PEG) linked dolastatinderivatives.

Such amino acids include amino acids having the structure of Formula(XXXVII-A) or (XXXVII-B):

wherein:

-   -   A is optional, and when present is lower alkylene, substituted        lower alkylene, lower cycloalkylene, substituted lower        cycloalkylene, lower alkenylene, substituted lower alkenylene,        alkynylene, lower heteroalkylene, substituted heteroalkylene,        lower heterocycloalkylene, substituted lower        heterocycloalkylene, arylene, substituted arylene,        heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;    -   B is optional, and when present is a linker linked at one end to        a diamine containing moiety, the linker selected from the group        consisting of lower alkylene, substituted lower alkylene, lower        alkenylene, substituted lower alkenylene, lower heteroalkylene,        substituted lower heteroalkylene, —O-(alkylene or substituted        alkylene)-, —S-(alkylene or substituted alkylene)-, —C(O)R″—,        —C(O)R″—, —S(O)_(k)(alkylene or substituted alkylene)-, where k        is 1, 2, or 3, —C(O)-(alkylene or substituted alkylene)-,        —C(S)-(alkylene or substituted alkylene)-, —NR″-(alkylene or        substituted alkylene)-, —CON(R″)-(alkylene or substituted        alkylene)-, —CSN(R″)-(alkylene or substituted alkylene)-, and        —N(R″)CO-(alkylene or substituted alkylene)-, where each R″ is        independently H, alkyl, or substituted alkyl;    -   K is

-   -    where:        -   R₈ and R₉ are independently selected from H, alkyl,            substituted alkyl, cycloalkyl, substituted cycloalkyl, or            amine protecting group;        -   T₁ is a bond, optionally substituted C₁-C₄ alkylene,            optionally substituted C₁-C₄ alkenylene, or optionally            substituted heteroalkyl;        -   T₂ is optionally substituted C₁-C₄ alkylene, optionally            substituted C₁-C₄ alkenylene, optionally substituted            heteroalkyl, optionally substituted aryl, or optionally            substituted heteroaryl;        -   wherein each optional substituents is independently selected            from lower alkyl, substituted lower alkyl, lower cycloalkyl,            substituted lower cycloalkyl, lower alkenyl, substituted            lower alkenyl, alkynyl, lower heteroalkyl, substituted            heteroalkyl, lower heterocycloalkyl, substituted lower            heterocycloallyl, aryl, substituted aryl, heteroaryl,            substituted heteroaryl, alkaryl, substituted alkaryl,            aralkyl, or substituted aralkyl;    -   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted        cycloalkyl;    -   R₁ is H, an amino protecting group, resin, amino acid,        polypeptide, or polynucleotide; and    -   R₂ is OH, an ester protecting group, resin, amino acid,        polypeptide, or polynucleotide;    -   each of R₃ and R₄ is independently H, halogen, lower alkyl, or        substituted lower alkyl, or R₃ and R₄ or two R₃ groups        optionally form a cycloalkyl or a heterocycloalkyl;        -   or the -A-B—K—R groups together form a bicyclic or tricyclic            cycloalkyl or heterocycloalkyl comprising at least one            diamine group, protected diamine group or masked diamine            group;        -   or the —B—K—R groups together form a bicyclic or tricyclic            cycloalkyl or cycloaryl or heterocycloalkyl comprising at            least one diamine group, protected diamine group or masked            diamine group;        -   or the —K—R group together forms a monocyclic or bicyclic            cycloalkyl or heterocycloalkyl comprising at least one            diamine group, protected diamine group or masked diamine            group;            wherein at least one amine group on -A-B—K—R is optionally a            protected amine.

In one aspect are compounds comprising the structures 1 or 2:

wherein:

-   -   A is optional, and when present is lower alkylene, substituted        lower alkylene, lower cycloalkylene, substituted lower        cycloalkylene, lower alkenylene, substituted lower alkenylene,        alkynylene, lower heteroalkylene, substituted heteroalkylene,        lower heterocycloalkylene, substituted lower        heterocycloalkylene, arylene, substituted arylene,        heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;    -   B is optional, and when present is a linker linked at one end to        a diamine containing moiety, the linker selected from the group        consisting of lower alkylene, substituted lower alkylene, lower        alkenylene, substituted lower alkenylene, lower heteroalkylene,        substituted lower heteroalkylene, —O-(alkylene or substituted        alkylene)-, —S-(alkylene or substituted alkylene)-, —C(O)R″—,        —S(O)_(k)(alkylene or substituted alkylene)-, where k is 1, 2,        or 3, —C(O)-(alkylene or substituted alkylene)-, —C(S)-(alkylene        or substituted alkylene)-, —NR″-(alkylene or substituted        alkylene)-, —CON(R″)-(alkylene or substituted alkylene)-,        —CSN(R″)-(alkylene or substituted alkylene)-, and        —N(R″)CO-(alkylene or substituted alkylene)-, where each R″ is        independently H, alkyl, or substituted alkyl;    -   T₁ is a bond or CH₂; and T₂ is CH;    -   wherein each optional substituents is independently selected        from lower alkyl, substituted lower alkyl, lower cycloalkyl,        substituted lower cycloalkyl, lower alkenyl, substituted lower        alkenyl, alkynyl, lower heteroalkyl, substituted heteroalkyl,        lower heterocycloalkyl, substituted lower heterocycloalkyl,        aryl, substituted aryl, heteroaryl, substituted heteroaryl,        alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl;    -   R₁ is H, an amino protecting group, resin, amino acid,        polypeptide, or polynucleotide; and    -   R₂ is OH, an ester protecting group, resin, amino acid,        polypeptide, or polynucleotide;    -   each of R₃ and R₄ is independently H, halogen, lower alkyl, or        substituted lower alkyl, or R₃ and R₄ or two R₃ groups        optionally form a cycloalkyl or a heterocycloalkyl;    -   or the -A-B-diamine containing moiety together form a bicyclic        cycloalkyl or heterocycloalkyl comprising at least one diamine        group, protected diamine group or masked diamine group;    -   or the —B-diamine containing moiety groups together form a        bicyclic or tricyclic cycloalkyl or cycloaryl or        heterocycloalkyl comprising at least one diamine group,        protected diamine group or masked diamine group;    -   wherein at least one amine group on -A-B-diamine containing        moiety is optionally a protected amine;    -   or an active metabolite, salt, or a pharmaceutically acceptable        prodrug or solvate thereof.

The following non-limiting examples of amino acids having the structureof Formula (XXXVII) are included:

Such non-natural amino acids may also be in the form of a salt or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and/or optionally posttranslationally modified.

In certain embodiments, compounds of Formula (XXXVII) are stable inaqueous solution for at least 1 month under mildly acidic conditions. Incertain embodiments, compounds of Formula (XXXVII) are stable for atleast 2 weeks under mildly acidic conditions. In certain embodiments,compound of Formula (XXXVII) are stable for at least 5 days under mildlyacidic conditions. In certain embodiments, such acidic conditions are pHabout 2 to about 8.

In certain embodiments of compounds of Formula (XXXVII), B is loweralkylene, substituted lower alkylene, O-(alkylene or substitutedalkylene)-, C(R′)═NN(R′)—, —N(R′)CO—, C(O)—, —C(R′)═N—, C(O)-(alkyleneor substituted alkylene)-, CON(R′)alkylene or substituted alkylene)-,—S(alkylene or substituted alkylene)-, —S(O)(alkylene or substitutedalkylene)-, or —S(O)₂(alkylene or substituted alkylene)-. In certainembodiments of compounds of Formula (XXXVII), B is —O(CH₂)—, —CH═N—,CH═NNH—, —NHCH₂—, —NHCO—, C(O)—, C(O)(CH₂)—, CONH(CH₂)—, —SCH₂—,—S(—O)CH₂—, or —S(O)₂CH₂—. In certain embodiments of compounds ofFormula (XXXVII), R is C₁₋₆alkyl or cycloalkyl. In certain embodimentsof compounds of Formula (XXXVII) R is —CH₃, —CH(CH3)₂, or cyclopropyl.In certain embodiments of compounds of Formula (XXXVII), R₁ is H,tert-butyloxycarbonyl (Boc), 9-Fluorenylmethoxycarbonyl (Fmoc),N-acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). Incertain embodiments of compounds of Formula (XXXVII), R₁ is a resin,amino acid, polypeptide, or polynucleotide. In certain embodiments ofcompounds of Formula (XXXVII), R₁ is an antibody, antibody fragment ormonoclonal antibody. In certain embodiments of compounds of Formula(XXXVII), R₂ is OH, O-methyl, O-ethyl, or O-t-butyl. In certainembodiments of compounds of Formula (XXXVII), R₂ is a resin, at leastone amino acid, polypeptide, or polynucleotide. In certain embodimentsof compounds of Formula (XXXVII), R₂ is an antibody, antibody fragmentor monoclonal antibody.

The following non-limiting examples of amino acids having the structureof Formula (XXXVII) are also included:

Non-limiting examples of protected amino acids having the structure ofFormula (XXXVII) include:

F. Structure and Synthesis of Non-Natural Amino Acids: Aromatic Amines

Non-natural amino acids with nucleophilic reactive groups, such as, byway of example only, an aromatic amine group (including secondary andtertiary amine groups), a masked aromatic amine group (which can bereadily converted into a aromatic amine group), or a protected aromaticamine group (which has reactivity similar to an aromatic amine groupupon deprotection) allow for a variety of reactions to link moleculesvia various reactions, including but not limited to, reductivealkylation reactions with aldehyde containing dolastatin linkerderivatives. Such aromatic amine containing non-natural amino acidsinclude amino acids having the structure of Formula (XXXXXXV):

wherein:

is selected from the group consisting of a monocyclic aryl ring, abicyclic aryl ring, a multicyclic aryl ring, a monocyclic heteroarylring, a bicyclic heteroaryl ring, and a multicyclic heteroaryl ring;

-   A is independently CR_(a), or N;-   B is independently CR_(a), N, O, or S;-   each R_(a) is independently selected from the group consisting of H,    halogen, alkyl, —NO₂, —CN, substituted alkyl, —N(R′)₂, —C(O)_(k)R′,    —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where k is 1, 2, or 3; and n is    0, 1, 2, 3, 4, 5, or 6;-   R₁ is H, an amino protecting group, resin, at least one amino acid,    polypeptide, or polynucleotide; and-   R₂ is OH, an ester protecting group, resin, at least one amino acid,    polypeptide, or polynucleotide;-   each of R₃ and R₄ is independently H, halogen, lower alkyl, or    substituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally    form a cycloalkyl or a heterocycloalkyl;-   M is H or —CH₂R₅; or the M-N—C(R₅) moiety may form a 4 to 7 membered    ring structure;-   R₅ is alkyl, substituted alkyl, alkenyl, substituted alkenyl,    alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,    alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide,    substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl,    aryl, substituted aryl, heteroaryl, substituted heteroaryl,    heterocycle, substituted heterocycle, alkaryl, substituted alkaryl,    aralkyl, substituted aralkyl, —C(O)R″, —C(O)OR″, —C(O)N(R″)₂,    —C(O)NHCH(R″)₂, -(alkylene or substituted alkylene)-N(R″)₂,    -(alkenylene or substituted alkenylene)-N(R″)₂, -(alkylene or    substituted alkylene)-(aryl or substituted aryl), -(alkenylene or    substituted alkenylene)-(aryl or substituted aryl), -(alkylene or    substituted alkylene)-ON(R″)₂, -(alkylene or substituted    alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or    substituted aryl), wherein each R″ is independently hydrogen, alkyl,    substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted    alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,    heterocycle, substituted heterocycle, alkaryl, substituted alkaryl,    aralkyl, substituted aralkyl, or —C(O)OR′;-   or two R₅ groups optionally form a cycloalkyl or a heterocycloalkyl;-   or R₅ and any R_(a) optionally form a cycloalkyl or a    heterocycloalkyl; and-   each R′ is independently H, alkyl, or substituted alkyl.    Such non-natural amino acids may also be in the form of a salt, or    may be incorporated into a non-natural amino acid polypeptide,    polymer, polysaccharide, or a polynucleotide and optionally    reductively alkylated.    The structure

(as presented in all examples herein) does not present the relativeorientations of “A,” “B,” “NH-M” and “R_(a)”; rather these four featuresof this structure may be oriented in any chemically-sound manner (alongwith other features of this structure), as illustrated by exampleherein.

Non-natural amino acids containing an aromatic amine moiety having thestructure of Formula (A) include non-natural amino acids having thestructures:

wherein, each A′ is independently selected from CR_(a), N, or

and up to two A′ may be

with the remaining A′ selected from CR_(a), orSuch non-natural amino acids may also be in the form of a salt, or maybe incorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally reductivelyalkylated.

Non-limiting examples of non-natural amino acids containing an aromaticamine moiety having the structure of Formula (XXXXXXV) includenon-natural amino acids having the structure of Formula (XXXXXXVI), andFormula (XXXXXXVII),

wherein; G is an amine protecting group, including, but not limited to,

Such non-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally reductivelyalkylated.

Non-natural amino acids containing an aromatic amine moiety have thefollowing structures:

-   wherein each R_(a) is independently selected from the group    consisting of H, halogen, alkyl, —NO₂, —CN, substituted alkyl,    —N(R′)₂, —C(O)_(k)R′, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where k is    1, 2, or 3;-   M is H or —CH₂R₅; or the M-N—C(R₅) moiety may form a 4 to 7 membered    ring structure;-   R₁ is H, an amino protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   R₂ is OH, an ester protecting group, resin, amino acid, polypeptide,    or polynucleotide;-   R₅ is alkyl, substituted alkyl, alkenyl, substituted alkenyl,    alkynyl, substituted alkynyl, alkoxy, substituted alkoxy,    alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide,    substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl,    aryl, substituted aryl, heteroaryl, substituted heteroaryl,    heterocycle, substituted heterocycle, alkaryl, substituted alkaryl,    aralkyl, substituted aralkyl, —C(O)R″, —C(O)OR″, —C(O)N(R″)₂,    —C(O)NHCH(R″)₂, -(alkylene or substituted alkylene)-N(R″)₂,    -(alkenylene or substituted alkenylene)-N(R″)₂, -(alkylene or    substituted alkylene)-(aryl or substituted aryl), -(alkenylene or    substituted alkenylene)-(aryl or substituted aryl), -(alkylene or    substituted alkylene)-ON(R″)₂, -(alkylene or substituted    alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or    substituted aryl), wherein each R″ is independently hydrogen, alkyl,    substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted    alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl,    heterocycle, substituted heterocycle, alkaryl, substituted alkaryl,    aralkyl, substituted aralkyl, or —C(O)OR′;    or R₅ and any R_(a) optionally form a cycloalkyl or a    heterocycloalkyl; and    each R′ is independently H, alkyl, or substituted alkyl. Such    non-natural amino acids may also be in the form of a salt, or may be    incorporated into a non-natural amino acid polypeptide, polymer,    polysaccharide, or a polynucleotide.

Such non-natural amino acids of Formula (XXXXXXV) may be formed byreduction of protected or masked amine moieties on the aromatic moietyof a non-natural amino acid. Such protected or masked amine moietiesinclude, but are not limited to, imines, hydrazines, nitro, or azidesubstituents. The reducing agents used to reduce such protected ormasked amine moieties include, but are not limited to, TCEP, Na₂S,Na₂S₂O₄, LiAH₄, NaBH₄ or NaBCNH₃.

V. Non-Natural Amino Acid Linked Dolastatin Derivatives

In another aspect described herein are methods, strategies andtechniques for incorporating at least one such dolastatin linkerderivatives into a non-natural amino acid. Also included with thisaspect are methods for producing, purifying, characterizing and usingsuch dolastatin linker derivatives containing at least one suchnon-natural amino acid. Also included with this aspect are compositionsof and methods for producing, purifying, characterizing and usingoligonucleotides (including DNA and RNA) that can be used to produce, atleast in part, a dolastatin linker derivative containing at least onenon-natural amino acid. Also included with this aspect are compositionsof and methods for producing, purifying, characterizing and using cellsthat can express such oligonucleotides that can be used to produce, atleast in part, a dolastatin linker derivative containing at least onenon-natural amino acid.

Thus, dolastatin linker derivatives comprising at least one non-naturalamino acid or modified non-natural amino acid with a carbonyl,dicarbonyl, alkyne, cycloalkyne, azide, oxime or hydroxylamine group areprovided and described herein. In certain embodiments, dolastatin linkerderivatives with at least one non-natural amino acid or modifiednon-natural amino acid with a carbonyl, dicarbonyl, alkyne, cycloalkyne,azide, oxime or hydroxylamine group include at least onepost-translational modification at some position on the polypeptide. Insome embodiments the co-translational or post-translational modificationoccurs via the cellular machinery (e.g., glycosylation, acetylation,acylation, lipid-modification, palmitoylation, palmitate addition,phosphorylation, glycolipid-linkage modification, and the like), in manyinstances, such cellular-machinery-based co-translational orpost-translational modifications occur at the naturally occurring aminoacid sites on the polypeptide, however, in certain embodiments, thecellular-machinery-based co-translational or post-translationalmodifications occur on the non-natural amino acid site(s) on thepolypeptide.

In other embodiments, the post-translational modification does notutilize the cellular machinery, but the functionality is insteadprovided by attachment of a molecule (a polymer; a water-solublepolymer; a derivative of polyethylene glycol; a second protein orpolypeptide or polypeptide analog; an antibody or antibody fragment; andany combination thereof) comprising a second reactive group to the atleast one non-natural amino acid comprising a first reactive group(including but not limited to, non-natural amino acid containing aketone, aldehyde, acetal, hemiacetal, alkyne, cycloalkyne, azide, oxime,or hydroxylamine functional group) utilizing chemistry methodologydescribed herein, or others suitable for the particular reactive groups.In certain embodiments, the co-translational or post-translationalmodification is made in vivo in a eukaryotic cell or in a non-eukaryoticcell. In certain embodiments, the post-translational modification ismade in vitro not utilizing the cellular machinery. Also included withthis aspect are methods for producing, purifying, characterizing andusing such dolastatin linker derivatives containing at least one suchco-translationally or post-translationally modified non-natural aminoacids.

Also included within the scope of the methods, compositions, strategiesand techniques described herein are reagents capable of reacting with adolastatin linker derivative (containing a carbonyl or dicarbonyl group,oxime group, alkyne, cycloalkyne, azide, hydroxylamine group, or maskedor protected forms thereof) that is part of a polypeptide so as toproduce any of the aforementioned post-translational modifications. Incertain embodiments, the resulting post-translationally modifieddolastatin linker derivative will contain at least one oxime group; theresulting modified oxime-containing dolastatin linker derivative mayundergo subsequent modification reactions. Also included with thisaspect are methods for producing, purifying, characterizing and usingsuch reagents that are capable of any such post-translationalmodifications of such dolastatin linker derivative(s).

In certain embodiments, the polypeptide or non-natural amino acid linkeddolastatin derivative includes at least one co-translational orpost-translational modification that is made in vivo by one host cell,where the post-translational modification is not normally made byanother host cell type. In certain embodiments, the polypeptide includesat least one co-translational or post-translational modification that ismade in vivo by a eukaryotic cell, where the co-translational orpost-translational modification is not normally made by a non-eukaryoticcell. Examples of such co-translational or post-translationalmodifications include, but are not limited to, glycosylation,acetylation, acylation, lipid-modification, palmitoylation, palmitateaddition, phosphorylation, glycolipid-linkage modification, and thelike. In one embodiment, the co-translational or post-translationalmodification comprises attachment of an oligosaccharide to an asparagineby a GlcNAc-asparagine linkage (including but not limited to, where theoligosaccharide comprises (GlcNAc-Man)₂-Man-GlcNAc-GlcNAc, and thelike). In another embodiment, the co-translational or post-translationalmodification comprises attachment of an oligosaccharide (including butnot limited to, Gal-GalNAc, Gal-GlcNAc, etc.) to a serine or threonineby a GalNAc-serine, a to GalNAc-threonine, a GlcNAc-serine, or aGlcNAc-threonine linkage. In certain embodiments, a protein orpolypeptide can comprise a secretion or localization sequence, anepitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, and/or thelike. Also included with this aspect are methods for producing,purifying, characterizing and using such polypeptides containing atleast one such co-translational or post-translational modification. Inother embodiments, the glycosylated non-natural amino acid polypeptideis produced in a non-glycosylated form. Such a non-glycosylated form ofa glycosylated non-natural amino acid may be produced by methods thatinclude chemical or enzymatic removal of oligosaccharide groups from anisolated or substantially purified or unpurified glycosylatednon-natural amino acid polypeptide; production of the non-natural aminoacid in a host that does not glycosylate such a non-natural amino acidpolypeptide (such a host including, prokaryotes or eukaryotes engineeredor mutated to not glycosylate such a polypeptide), the introduction of aglycosylation inhibitor into the cell culture medium in which such anon-natural amino acid polypeptide is being produced by a eukaryote thatnormally would glycosylate such a polypeptide, or a combination of anysuch methods. Also described herein are such non-glycosylated forms ofnormally-glycosylated non-natural amino acid polypeptides (bynormally-glycosylated is meant a polypeptide that would be glycosylatedwhen produced under conditions in which naturally-occurring polypeptidesare glycosylated). Of course, such non-glycosylated forms ofnormally-glycosylated non-natural amino acid polypeptides (or indeed anypolypeptide described herein) may be in an unpurified form, asubstantially purified form, or in an isolated form.

In certain embodiments, the non-natural amino acid polypeptide includesat least one post-translational modification that is made in thepresence of an accelerant, wherein the post-translational modificationis stoichiometric, stoichiometric-like, or near-stoichiometric. In otherembodiments the polypeptide is contacted with a reagent of Formula (XIX)in the presence of an accelerant. In other embodiments the accelerant isselected from the group consisting of:

A. Chemical Synthesis of Non-Natural Amino Acid Linked DolastatinDerivatives: Oxime-Containing Linked Dolastatin Derivatives

Non-natural amino acid dolastatin linked derivatives containing an oximegroup allow for reaction with a variety of reagents that contain certainreactive carbonyl- or dicarbonyl-groups (including but not limited to,ketones, aldehydes, or other groups with similar reactivity) to form newnon-natural amino acids comprising a new oxime group. Such an oximeexchange reaction allows for the further functionalization of dolastatinlinked derivatives. Further, the original dolastatin linked derivativecontaining an oxime group may be useful in their own right as long asthe oxime linkage is stable under conditions necessary to incorporatethe amino acid into a polypeptide (e.g., the in vivo, in vitro andchemical synthetic methods described herein).

Thus, in certain embodiments described herein are non-natural amino aciddolastatin linked derivatives with sidechains comprising an oxime group,an oxime-like group (which has reactivity similar to an oxime group andis structurally similar to an oxime group), a masked oxime group (whichcan be readily converted into an oxime group), or a protected oximegroup (which has reactivity similar to an oxime group upondeprotection).

Such non-natural amino acid dolastatin linked derivatives includedolastatin linked derivatives having the structure of Formula (VIII) or(IX):

wherein:

-   -   A is optional, and when present is lower alkylene, substituted        lower alkylene, lower cycloalkylene, substituted lower        cycloalkylene, lower alkenylene, substituted lower alkenylene,        alkynylene, lower heteroalkylene, substituted heteroalkylene,        lower heterocycloalkylene, substituted lower        heterocycloalkylene, arylene, substituted arylene,        heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;    -   B is optional, and when present is a linker selected from the        group consisting of lower alkylene, substituted lower alkylene,        lower alkenylene, substituted lower alkenylene, lower        heteroalkylene, substituted lower heteroalkylene, —O—,        —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or        substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or 3,        —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,        —C(O)-(alkylene or substituted alkylene)-, —C(S)—,        —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,        —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,        —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,        —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene        or substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,        —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—,        —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═,        —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ is        independently H, alkyl, or substituted alkyl;    -   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted        cycloalkyl;    -   R₁ is H, an amino protecting group, resin, at least one amino        acid, polypeptide, or polynucleotide;    -   R₂ is OH, an ester protecting group, resin, at least one amino        acid, polypeptide, or polynucleotide;    -   R₃ and R₄ are each independently H, halogen, lower alkyl, or        substituted lower alkyl, or R₃ and R₄ or two R₃ groups        optionally form a cycloalkyl or a heterocycloalkyl;    -   Z has the structure of:

-   -   -   R₅ is H, COR₈, C₁-C₆alkyl, or thiazole;            -   R₈ is OH        -   R₆ is OH or H;        -   Ar is phenyl or pyridine;

    -   R₇ is C₁-C₆alkyl or hydrogen;

    -   L is a linker selected from the group consisting of -alkylene-,        -alkylene-C(O)—, -(alkylene-O)_(n)-alkylene-,        -(alkylene-O)_(n)-alkylene-C(O)—,        -(alkylene-O)_(n)—(CH₂)_(n′)—NHC(O)(CH₂)_(n″)—C(Me)₂-S—S—CH₂)_(n′″)—NHC(O)-(alkylene-O)_(n″″)-alkylene-,        -(alkylene-O)_(n)-alkylene-W—, -alkylene-C(O)—W—,        -alkylene-O)_(n)-alkylene-U-alkylene-C(O)—, and        -(alkylene-O)_(n)-alkylene-U-alkylene-;        -   W has the structure of:

-   -   -   U has the structure of:

and

-   -   each n, n′, n′, n′″ and n″″ are independently integers greater        than or equal to one; or an active metabolite, or a        pharmaceutically acceptable prodrug or solvate thereof.

In certain embodiments of compounds of Formula (VIII) and (IX), R₅ isthiazole. In certain embodiments of compounds of Formula (VIII) and(IX), R₆ is H. In certain embodiments of compounds of Formula (VIII) and(IX), Ar is phenyl. In certain embodiments of compounds of Formula(VIII) and (IX), R₇ is methyl. In certain embodiments of compounds ofFormula (VIII) and (IX), n is an integer from 0 to 20. In certainembodiments of compounds of Formula (VIII) and (IX), n is an integerfrom 0 to 10. In certain embodiments of compounds of Formula (VIII) and(IX), n is an integer from 0 to 5.

In certain embodiments of compounds of Formula (VIII) and (IX), R₅ isthiazole. In certain embodiments of compounds of Formula (VIII) and(IX), R₅ is hydrogen. In certain embodiments of compounds of Formula(VIII) and (IX), R₅ is methyl, ethyl, propyl, iso-propyl, butyl,iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl. In certainembodiments of compounds of Formula (VIII) and (IX), R₅ is—NH-(alkylene-O)_(n)—NH₂, wherein alkylene is —CH₂—, —CH₂CH₂—,—CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, or—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—. In certain embodiments ofFormula (VIII) and (IX), alkylene is methylene, ethylene, propylene,butylenes, pentylene, hexylene, or heptylene.

In certain embodiments of compounds of Formula (VIII) and (IX), R₅ is—NH-(alkylene-O)_(n)—NH₂, wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100.

In certain embodiments of compounds of Formula (VIII) and (IX), R₆ is H.

In certain embodiments of compounds of Formula (VIII) and (IX), Ar isphenyl.

In certain embodiments of compounds of Formula (VIII) and (IX), R₇ ismethyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl,tert-butyl, pentyl, or hexyl. In certain embodiments of compounds ofFormula (VIII) and (IX), R₇ is hydrogen.

In certain embodiments of compounds of Formula (VIII) and (IX), each Lis independently a cleavable linker or non-cleavable linker. In certainembodiments of compounds of Formula (VIII) and (IX), each L isindependently a oligo(ethylene glycol) derivatized linker.

In certain embodiments of compounds of Formula (VIII) and (IX), eachalkylene, alkylene′, alkylene″, and alkylene′″ independently is —CH₂—,—CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂Cl₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,or —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—. In certain embodiments ofcompounds of Formula (VIII) and (IX), alkylene is methylene, ethylene,propylene, butylenes, pentylene, hexylene, or heptylene.

In certain embodiments of compounds of Formula (VIII) and (IX), each n,n′, n″, n′″, and n″″ independently is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100.

In certain embodiments of compounds of Formula (VIII) or (IX), R₁ is apolypeptide. In certain embodiments of compounds of Formula (VIII) or(IX), R₂ is a polypeptide. In certain embodiments of compounds ofFormula (VIII) or (IX), the polypeptide is an antibody. In certainembodiments of compounds of Formula (VIII) or (IX), the antibody isherceptin.

Such non-natural amino acid dolastatin linked derivatives includedolastatin linked derivatives having the structure of Formula (X), (XI),(XII) or (XIII):

wherein:

-   -   A is optional, and when present is lower alkylene, substituted        lower alkylene, lower cycloalkylene, substituted lower        cycloalkylene, lower alkenylene, substituted lower alkenylene,        alkynylene, lower heteroalkylene, substituted heteroalkylene,        lower heterocycloalkylene, substituted lower        heterocycloalkylene, arylene, substituted arylene,        heteroarylene, substituted heteroarylene, alkarylene,        substituted alkarylene, aralkylene, or substituted aralkylene;    -   B is optional, and when present is a linker selected from the        group consisting of lower alkylene, substituted lower alkylene,        lower alkenylene, substituted lower alkenylene, lower        heteroalkylene, substituted lower heteroalkylene, —O—,        —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or        substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or 3,        —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,        —C(O)-(alkylene or substituted alkylene)-, —C(S)—,        —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,        —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,        —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,        —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene        or substituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—,        —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—,        —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═,        —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ is        independently H, alkyl, or substituted alkyl;    -   R is H, alkyl, substituted alkyl, cycloalkyl, or substituted        cycloalkyl;    -   R₁ is H, an amino protecting group, resin, at least one amino        acid, polypeptide, or polynucleotide;    -   R₂ is OH, an ester protecting group, resin, at least one amino        acid, polypeptide, or polynucleotide;    -   R₃ and R₄ are each independently H, halogen, lower alkyl, or        substituted lower alkyl, or R₃ and R₄ or two R₃ groups        optionally form a cycloalkyl or a heterocycloalkyl;    -   Z has the structure of:

-   -   -   R₅ is H, CO₂H, C₁-C₆alkyl, or thiazole;        -   R₆ is OH or H;        -   Ar is phenyl or pyridine;

    -   R₇ is C₁-C₆alkyl or hydrogen;

    -   L₁, L₂, L₃, and L₄ are each linkers independently selected from        the group consisting of a bond, -alkylene-,        -(alkylene-O)_(n)-alkylene-J-,        -alkylene′-J-(alkylene-O)_(n)-alkylene-,        -J-(alkylene-O)_(n)-alkylene-,        -(alkylene-O)_(n)-alkylene-J-(alkylene-O)_(n)′-alkylene-J′-,        -(alkylene-O)_(n)-alkylene-J-alkylene′-, —W—, -alkylene-W—,        alkylene′-J-(alkylene-NMe)_(n)-alkylene-W—,        -J-(alkylene-NMe)_(n)-alkylene-W—,        -J-alkylene-NMe-alkylene′-NMe-alkylene″-W—, and        -alkylene-J-alkylene′-NMe-alkylene″-NMe-alkylene′″-W—;        -   W has the structure of:

-   -   -   each J and J′ independently have the structure of:

-   -    and    -   each n and n′ are independently integers greater than or equal        to one.

In certain embodiments of compounds of Formula (X), (XI), (XII) or(XIII), R₅ is thiazole or carboxylic acid. In certain embodiments ofcompounds of Formula (X), (XI), (XII) or (XIII), R₆ is H. In certainembodiments of compounds of Formula (X), (XI), (XII) or (XIII), Ar isphenyl. In certain embodiments of compounds of Formula (X), (XI), (XII)or (XIII), R₇ is methyl. In certain embodiments of compounds of Formula(X), (XI), (XII) or (XIII), n and n′ are integers from 0 to 20. Incertain embodiments of compounds of Formula (X), (XI), (XII) or (XIII),n and n′ are integers from 0 to 10. In certain embodiments of compoundsof Formula (X), (XI), (XII) or (XIII), n and n′ are integers from 0 to5.

In certain embodiments of compounds of Formula (X), (XI), (XII) or(XIII), R₅ is thiazole. In certain embodiments of compounds of Formula(X), (XI), (XII) or (XIII), R₅ is hydrogen. In certain embodiments ofcompounds of Formula (X), (XI), (XII) or (XIII), R₅ is methyl, ethyl,propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, orhexyl. In certain embodiments of compounds of Formula (X), (XI), (XII)or (XIII), R₅ is —NH-(alkylene-O)_(n)—NH₂, wherein alkylene is —CH₂—,—CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂—CH—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,or —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—. In certain embodiments ofFormula (X), (XI), (XII) or (XIII), alkylene is methylene, ethylene,propylene, butylenes, pentylene, hexylene, or heptylene.

In certain embodiments of compounds of Formula (X), (XI), (XII) or(XIII), R₅ is —NH-(alkylene-O)_(n)—NH₂, wherein n is 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100.

In certain embodiments of compounds of Formula (X), (XI), (XII) or(XIII), R₆ is H. In some embodiments of compounds of Formula (X), (XI),(XII) or (XIII), R₆ is hydroxy.

In certain embodiments of compounds of Formula (X), (XI), (XII) or(XIII), Ar is phenyl.

In certain embodiments of compounds of Formula (X), (XI), (XII) or(XIII), R₇ is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyliso-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments ofcompounds of Formula (X), (XI), (XII) or (XIII), R₇ is hydrogen.

In certain embodiments of compounds of Formula (X), (XI), (XII) or(XIII), each L₁, L₂, L₃, and L₄ is independently a cleavable linker ornon-cleavable linker. In certain embodiments of compounds of Formula(X), (XI), (XII) or (XIII), each L₁, L₂, L₃, and L₄ is independently aoligo(ethylene glycol) derivatized linker.

In certain embodiments of compounds of Formula (X), (XI), (XII) or(XIII), each alkylene, alkylene′, alkylene″, and alkylene′″independently is —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂,—CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,or —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—. In certain embodiments ofcompounds of Formula (X), (XI), (XII) or (XIII), alkylene is methylene,ethylene, propylene, butylenes, pentylene, hexylene, or heptylene.

In certain embodiments of compounds of Formula (X), (XI), (XII) or(XIII), each n and n′ independently is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100.

In certain embodiments of compounds of Formula (X), (XI), (XII) or(XIII), R₁ is a polypeptide. In certain embodiments of compounds ofFormula (X), (XI), (XII) or (XIII), R₂ is a polypeptide. In certainembodiments of compounds of Formula (X), (XI), (XII) or (XIII), thepolypeptide is an antibody. In certain embodiments of compounds ofFormula (X), (XI), (XII) or (XIII), the antibody is herceptin.

In certain embodiments, compounds of Formula (X), (XI), (XII) or (XIII)are stable in aqueous solution for at least 1 month under mildly acidicconditions. In certain embodiments, compounds of Formula (X), (XI),(XII) or (XIII) are stable for at least 2 weeks under mildly acidicconditions. In certain embodiments, compound of Formula (X), (XI), (XII)or (XIII) are stable for at least 5 days under mildly acidic conditions.In certain embodiments, such acidic conditions are pH 2 to 8. Suchnon-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

Oxime-based non-natural amino acids may be synthesized by methodsalready described in the art, or by methods described herein, including:(a) reaction of a hydroxylamine-containing non-natural amino acid with acarbonyl- or dicarbonyl-containing reagent; (b) reaction of a carbonyl-or dicarbonyl-containing non-natural amino acid with ahydroxylamine-containing reagent; or (c) reaction of an oxime-containingnon-natural amino acid with certain carbonyl- or dicarbonyl-containingreagents.

B. Chemical Structure and Synthesis of Non-Natural Amino Acid LinkedDolastatin Derivatives: Alkylated Aromatic Amine Linked DolastatinDerivatives

In one aspect are dolastatin linker derivatives for the chemicalderivatization of non-natural amino acids based upon the reactivity ofan aromatic amine group. In further or additional embodiments, at leastone of the aforementioned non-natural amino acids is incorporated into adolastatin linker derivative, that is, such embodiments are non-naturalamino acid linked dolastatin derivatives. In further or additionalembodiments, the dolastatin linker derivatives are functionalized ontheir sidechains such that their reaction with a derivatizingnon-natural amino acid generates an amine linkage. In further oradditional embodiments, the dolastatin linker derivatives are selectedfrom dolastatin linker derivatives having aromatic amine sidechains. Infurther or additional embodiments, the dolastatin linker derivativescomprise a masked sidechain, including a masked aromatic amine group. Infurther or additional embodiments, the non-natural amino acids areselected from amino acids having aromatic amine sidechains. In furtheror additional embodiments, the non-natural amino acids comprise a maskedsidechain, including a masked aromatic amine group.

In another aspect are carbonyl-substituted dolastatin linker derivativessuch as, by way of example, aldehydes, and ketones, for the productionof derivatized non-natural amino acid polypeptides based upon an aminelinkage. In a further embodiment are aldehyde-substituted dolastatinlinker derivatives used to derivatize aromatic amine-containingnon-natural amino acid polypeptides via the formation of an aminelinkage between the derivatizing dolastatin linker and the aromaticamine-containing non-natural amino acid polypeptide.

In further or additional embodiments, the non-natural amino acidscomprise aromatic amine sidechains where the aromatic amine is selectedfrom an aryl amine or a heteroaryl amine. In a further or additionalembodiment, the non-natural amino acids resemble a natural amino acid instructure but contain aromatic amine groups. In another or furtherembodiment the non-natural amino acids resemble phenylalanine ortyrosine (aromatic amino acids). In one embodiment, the non-naturalamino acids have properties that are distinct from those of the naturalamino acids. In one embodiment, such distinct properties are thechemical reactivity of the sidechain; in a further embodiment thisdistinct chemical reactivity permits the sidechain of the non-naturalamino acid to undergo a reaction while being a unit of a polypeptideeven though the sidechains of the naturally-occurring amino acid unitsin the same polypeptide do not undergo the aforementioned reaction. In afurther embodiment, the sidechain of the non-natural amino acid has achemistry orthogonal to those of the naturally-occurring amino acids. Ina further embodiment, the sidechain of the non-natural amino acidcomprises a nucleophile-containing moiety; in a further embodiment, thenucleophile-containing moiety on the sidechain of the non-natural aminoacid can undergo a reaction to generate an amine-linked derivatizeddolastatin. In a further embodiment, the sidechain of the non-naturalamino acid comprises an electrophile-containing moiety; in a furtherembodiment, the electrophile-containing moiety on the sidechain of thenon-natural amino acid can undergo nucleophilic attack to generate anamine-linked derivatized dolastatin. In any of the aforementionedembodiments in this paragraph, the non-natural amino acid may exist as aseparate molecule or may be incorporated into a polypeptide of anylength; if the latter, then the polypeptide may further incorporatenaturally-occurring or non-natural amino acids.

Modification of non-natural amino acids described herein using reductivealkylation or reductive amination reactions have any or all of thefollowing advantages. First, aromatic amines can be reductivelyalkylated with carbonyl-containing compounds, including aldehydes, andketones, in a pH range of about 4 to about 10 (and in certainembodiments in a pH range of about 4 to about 7) to generate substitutedamine, including secondary and tertiary amine, linkages. Second, underthese reaction conditions the chemistry is selective for non-naturalamino acids as the sidechains of naturally occurring amino acids areunreactive. This allows for site-specific derivatization of polypeptideswhich have incorporated non-natural amino acids containing aromaticamine moieties or protected aldehyde moieties, including, by way ofexample, recombinant proteins. Such derivatized polypeptides andproteins can thereby be prepared as defined homogeneous products. Third,the mild conditions needed to effect the reaction of an aromatic aminemoiety on an amino acid, which has been incorporated into a polypeptide,with an aldehyde-containing reagent generally do not irreversiblydestroy the tertiary structure of the polypeptide (excepting, of course,where the purpose of the reaction is to destroy such tertiarystructure). Similarly, the mild conditions needed to effect the reactionof an aldehyde moiety on an amino acid, which has been to incorporatedinto a polypeptide and deprotected, with an aromatic amine-containingreagent generally do not irreversibly destroy the tertiary structure ofthe polypeptide (excepting, of course, where the purpose of the reactionis to destroy such tertiary structure). Fourth, the reaction occursrapidly at room temperature, which allows the use of many types ofpolypeptides or reagents that would otherwise be unstable at highertemperatures. Fifth, the reaction occurs readily is aqueous conditions,again allowing use of polypeptides and reagents incompatible (to anyextent) with non-aqueous solutions. Six, the reaction occurs readilyeven when the ratio of polypeptide or amino acid to reagent isstoichiometric, stoichiometric-like, or near-stoichiometric, so that itis unnecessary to add excess reagent or polypeptide to obtain a usefulamount of reaction product. Seventh, the resulting amine can be producedregioselectively and/or regiospecifically, depending upon the design ofthe amine and carbonyl portions of the reactants. Finally, the reductivealkylation of aromatic amines with aldehyde-containing reagents, and thereductive amination of aldehydes with aromatic amine containingreagents, generates amine, including secondary and tertiary amine,linkages which are stable under biological conditions.

Non-natural amino acids with nucleophilic reactive groups, such as, byway of example only, an aromatic amine group (including secondary andtertiary amine groups), a masked aromatic amine group (which can bereadily converted into a aromatic amine group), or a protected aromaticamine group (which has reactivity similar to a aromatic amine group upondeprotection) allow for a variety of reactions to link molecules viavarious reactions, including but not limited to, reductive alkylationreactions with aldehyde containing dolastatin linked derivatives. Suchalkylated non-natural amino acid linked dolastatin derivatives includeamino acids having the structure of Formula (XXV), (XXVI), (XXVII),(XXVIII), (XXIX), or (XXX):

wherein:

-   -   Z has the structure of:

-   -   -   R₅ is H, CO₂H, C₁-C₆alkyl, or thiazole;        -   R₆ is OH or H;        -   Ar is phenyl or pyridine;

    -   R₁ is H, an amino protecting group, resin, at least one amino        acid, polypeptide, or polynucleotide;

    -   R₂ is OH, an ester protecting group, resin, at least one amino        acid, polypeptide, or polynucleotide;

    -   R₄ is H, halogen, lower alkyl, or substituted lower alkyl;

    -   R₇ is C₁-C₆alkyl or hydrogen;

    -   L, L₁, L₂, L₃, and L₄ are each linkers selected from the group        consisting of a bond, -alkylene-, -alkylene-C(O)—,        -(alkylene-O)-alkylene-, -(alkylene-O)_(n)-alkylene-C(O)—,        -(alkylene-O)—(CH₂)_(n′)—NHC(O)—(CH₂)_(n″)—C(Me)₂-S—S—(CH₂)_(n′″)—NHC(O)-(alkylene-O)_(n″″)-alkylene-,        -(alkylene-O)_(n)-alkylene-W—, -alkylene-C(O)—W—,        -(alkylene-O)_(n)-alkylene-J-,        -alkylene′-J-(alkylene-O)_(n)-alkylene-,        -(alkylene-O)_(n)-alkylene-J-alkylene′,        -J-(alkylene-O)_(n)-alkylene-,        -(alkylene-O)_(n)-alkylene-J-(alkylene-O)_(n′)-alkylene-J′—,        —W—, -alkylene-W—, alkylene′-J-(alkylene-NMe)_(n)-alkylene-W—,        and J-(alkylene-NMe)-alkylene-W—,        -(alkylene-O)_(n)-alkylene-U-alkylene-C(O)—,        -(alkylene-O)_(n)-alkylene-U-alkylene-;        -J-alkylene-NMe-alkylene′-NMe-alkylene″-W—, and        -alkylene-J-alkylene′-NMe-alkylene″-NMe-alkylene′″-W—;        -   W has the structure of:

-   -   -   -   U has the structure of:

-   -   -   each J and J′ independently have the structure of:

-   -   -   each n and n′ are independently integers greater than or            equal to one; and

    -   each R₁₆ is independently selected from the group consisting of        hydrogen, halogen, alkyl, NO₂, CN, and substituted alkyl.        Such alkylated non-natural amino acid linked dolastatin        derivatives may also be in the form of a salt, or may be        incorporated into a non-natural amino acid polypeptide, polymer,        polysaccharide, or a polynucleotide and optionally reductively        alkylated.

In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII),(XXVIII), (XXIX), or (XXX), R₅ is thiazole or carboxylic acid. Incertain embodiments of compounds of Formula (XXV), (XXVI), (XXVII),(XXVIII), (XXIX), or (XXX), R₆ is H. In certain embodiments of compoundsof Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), Ar isphenyl. In certain embodiments of compounds of Formula (XXV), (XXVI),(XXVII), (XXVIII), (XXIX), or (XXX), R₇ is methyl. In certainembodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII),(XXIX), or (XXX), n is an integer from 0 to 20. In certain embodimentsof compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or(XXX), n is an integer from 0 to 10. In certain embodiments of compoundsof Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX) or (XXIV),n is an integer from 0 to 5.

In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII),(XXVIII), (XXIX), or (XXX), R₅ is thiazole or carboxylic acid. Incertain embodiments of compounds of Formula (XXV), (XXVI), (XXVII),(XXVIII), (XXIX), or (XXX), R₅ is hydrogen. In certain embodiments ofcompounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX),R₅ is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl,tert-butyl, pentyl, or hexyl. In certain embodiments of compounds ofFormula ((XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R₅ is—NH-(alkylene-O)—NH₂, wherein alkylene is —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, or—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—. In certain embodiments ofFormula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), alkylene ismethylene, ethylene, propylene, butylenes, pentylene, hexylene, orheptylene.

In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII),(XXVIII), (XXIX), or (XXX), R₅ is —NH-(alkylene-O)_(n)—NH₂, wherein n is0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII),(XXVIII), (XXIX), or (XXX), R₆ is H. In some embodiments of compounds ofFormula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R₆ ishydroxy.

In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII),(XXVIII), (XXIX), or (XXX), Ar is phenyl.

In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII),(XXVIII), (XXIX), or (XXX), R₇ is methyl, ethyl, propyl, iso-propyl,butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In certainembodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII),(XXIX), or (XXX), R₇ is hydrogen.

In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII),(XXVIII), (XXIX), or (XXX), each L, L₁, L₂, L₃, and L₄ is independentlya cleavable linker or non-cleavable linker. In certain embodiments ofcompounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX),each L, L₁, L₂, L₃, and L₄ is independently a oligo(ethylene glycol)derivatized linker.

In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII),(XXVIII), (XXIX), or (XXX), each alkylene, alkylene′, alkylene″, andalkylene′″ independently is —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, or—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—. In certain embodiments ofcompounds of Formula (XIX), (XX), (XXI), (XXII), (XXIII) or (XXIV),alkylene is methylene, ethylene, propylene, butylenes, pentylene,hexylene, or heptylene.

In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII),(XXVIII), (XXIX), or (XXX), each n, n′, n″, n′″, and n″″ independentlyis 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII),(XXVIII), (XXIX), or (XXX), R₁ is a polypeptide. In certain embodimentsof compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or(XXX), R₂ is a polypeptide. In certain embodiments of compounds ofFormula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), thepolypeptide is an antibody. In certain embodiments of compounds ofFormula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), the antibodyis herceptin.

Compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX)may be formed by the reductive alkylation of aromatic amine compoundswith carbonyl containing reagents such as, by way of example, ketones,esters, thioesters, and aldehydes.

In some embodiments, the masked amine moieties of non-natural aminoacids contained in polypeptides are initially reduced to givenon-natural amino acids containing aromatic amine moieties incorporatedinto non-natural amino acid polypeptides. Such aromatic amine moietiesare then reductive alkylated with carbonyl-containing reagents describedabove to give polypeptides containing non-natural amino acids of Formula(XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX). Such reactions mayalso be applied to non-natural amino acids incorporated into syntheticpolymers, polysaccharides, or polynucleotides. Additionally, suchreactions may be applied to non-incorporated non-natural amino acids. Byway of example the reducing agent used to reduce masked amine moietiesincludes, but is not limited to, TCEP, Na₂S, Na₂S₂O₄, LiAlH₄, B₂H₆, andNaBH₄. By way of example only, reductive alkylation may occur in aqueousbuffers with a pH of about 4 to about 7 and using a mild reducing agent,such as, by way of example only, sodium cyanoborohydride (NaBCNH₃). Inaddition, other reducing agents may be used for reductive alkylationincluding, but not limited to, TCEP, Na₂S, Na₂S₂O₄, LiAlH₄, B₂H₆, andNaBH₄.

A non-limiting exemplary syntheses of non-natural amino acidpolypeptides containing amino acids of Formula (XXV), (XXVI), (XXVII),(XXVIII), (XXIX), or (XXX) by reductive alkylation of secondary aromaticamine moieties, contained in non-natural amino acids, withcarbonyl-containing reagents described above. Such reductive alkylationsgive polypeptides containing non-natural amino acids with tertiaryaromatic amine moieties. Such reactions may also be applied tonon-natural amino acids incorporated into synthetic polymers,polysaccharides, or polynucleotides. Additionally, such reactions may beapplied to non-incorporated non-natural amino acids. By way of exampleonly, reductive alkylation may occur in aqueous buffers with a pH ofabout 4 to about 7 and using a mild reducing agent, such as, by way ofexample only, sodium cyanoborohydride (NaBCNH₃). In addition, otherreducing agents may be used for reductive alkylation including, but notlimited to, TCEP, Na₂S, Na₂S₂O₄, LiAlH₄, B₂H₆, and NaBH₄.

C. Chemical Synthesis of Non-Natural Amino Acid Linked DolastatinDerivatives: Heteroaryl-Containing Linked Dolastatin Derivatives

In one aspect are non-natural amino acids for the chemicalderivatization of dolastatin linked derivatives based upon thereactivity of a dicarbonyl group, including a group containing at leastone ketone group, and/or at least one aldehyde groups, and/or at leastone ester group, and/or at least one carboxylic acid, and/or at leastone thioester group, and wherein the dicarbonyl group can be a1,2-dicarbonyl group, a 1,3-dicarbonyl group, or a 1,4-dicarbonyl group.In further or additional aspects are non-natural amino acids for thechemical derivatization of dolastatin linked derivatives based upon thereactivity of a diamine group, including a hydrazine group, an amidinegroup, an imine group, a 1,1-diamine group, a 1,2-diamine group, a1,3-diamine group, and a 1,4-diamine group. In further or additionalembodiments, at least one of the aforementioned non-natural amino acidsis incorporated into a dolastatin linked derivative, that is, suchembodiments are non-natural amino acid linked dolastatin derivatives. Infurther or additional embodiments, the non-natural amino acids arefunctionalized on their sidechains such that their reaction with aderivatizing molecule generates a linkage, including aheterocyclic-based linkage, including a nitrogen-containing heterocycle,and/or an aldol-based linkage. In further or additional embodiments arenon-natural amino acid polypeptides that can react with a derivatizingdolastatin linker to generate a non-natural amino acid linked dolastatinderivatives containing a linkage, including a heterocyclic-basedlinkage, including a nitrogen-containing heterocycle, and/or analdol-based linkage. In further or additional embodiments, thenon-natural amino acids are selected from amino acids having dicarbonyland/or diamine sidechains. In further or additional embodiments, thenon-natural amino acids comprise a masked sidechain, including a maskeddiamine group and/or a masked dicarbonyl group. In further or additionalembodiments, the non-natural amino acids comprise a group selected from:keto-amine (i.e., a group containing both a ketone and an amine);keto-alkyne (i.e., a group containing both a ketone and an alkyne); andan ene-dione (i.e., a group containing a dicarbonyl group and analkene).

In further or additional embodiments, the non-natural amino acidscomprise dicarbonyl sidechains where the carbonyl is selected from aketone, an aldehyde, a carboxylic acid, or an ester, including athioester. In another embodiment are non-natural amino acids containinga functional group that is capable of forming a heterocycle, including anitrogen-containing heterocycle, upon treatment with an appropriatelyfunctionalized reagent. In a further or additional embodiment, thenon-natural amino acids resemble a natural amino acid in structure butcontain one of the aforementioned functional groups. In another orfurther embodiment the non-natural amino acids resemble phenylalanine ortyrosine (aromatic amino acids); while in a separate embodiment, thenon-natural amino acids resemble alanine and leucine (hydrophobic aminoacids). In one embodiment, the non-natural amino acids have propertiesthat are distinct from those of the natural amino acids. In oneembodiment, such distinct properties are the chemical reactivity of thesidechain, in a further embodiment this distinct chemical reactivitypermits the sidechain of the non-natural amino acid to undergo areaction while being a unit of a polypeptide even though the sidechainsof the naturally-occurring amino acid units in the same polypeptide donot undergo the aforementioned reaction. In a further embodiment, thesidechain of the non-natural amino acid has a chemistry orthogonal tothose of the naturally-occurring amino acids. In a further embodiment,the sidechain of the non-natural amino acid comprises anelectrophile-containing moiety; in a further embodiment, theelectrophile-containing moiety on the sidechain of the non-natural aminoacid can undergo nucleophilic attack to generate aheterocycle-derivatized protein, including a nitrogen-containingheterocycle-derivatized protein. In any of the aforementionedembodiments in this paragraph, the non-natural amino acid may exist as aseparate molecule or may be incorporated into a polypeptide of anylength; if the latter, then the polypeptide may further incorporatenaturally-occurring or non-natural amino acids.

In another aspect are diamine-substituted molecules, wherein the diaminegroup is selected from a hydrazine, an amidine, an imine, a 1,1-diamine,a 1,2-diamine, a 1,3-diamine and a 1,4-diamine group, for the productionof derivatized non-natural amino acid linked dolastatin derivativesbased upon a heterocycle, including a nitrogen-containing heterocycle,linkage. In a further embodiment are diamine-substituted dolastatinderivatives used to derivatize dicarbonyl-containing non-natural aminoacid polypeptides via the formation of a heterocycle, including anitrogen-containing heterocycle, linkage between the derivatizingmolecule and the dicarbonyl-containing non-natural amino acidpolypeptide. In further embodiments the aforementioneddicarbonyl-containing non-natural amino acid polypeptides arediketone-containing non-natural amino acid polypeptides. In further oradditional embodiments, the dicarbonyl-containing non-natural aminoacids comprise sidechains where the carbonyl is selected from a ketone,an aldehyde, a carboxylic acid, or an ester, including a thioester. Infurther or additional embodiments, the diamine-substituted moleculescomprise a group selected from a desired functionality. In a furtherembodiment, the sidechain of the non-natural amino acid has a chemistryorthogonal to those of the naturally-occurring amino acids that allowsthe non-natural amino acid to react selectively with thediamine-substituted molecules. In a further embodiment, the sidechain ofthe non-natural amino acid comprises an electrophile-containing moietythat reacts selectively with the diamine-containing molecule; in afurther embodiment, the electrophile-containing moiety on the sidechainof the non-natural amino acid can undergo nucleophilic attack togenerate a heterocycle-derivatized protein, including anitrogen-containing heterocycle-derivatized protein. In a further aspectrelated to the embodiments described in this paragraph are the modifiednon-natural amino acid polypeptides that result from the reaction of thederivatizing molecule with the non-natural amino acid polypeptides.Further embodiments include any further modifications of the alreadymodified non-natural amino acid polypeptides.

In another aspect are dicarbonyl-substituted molecules for theproduction of derivatized non-natural amino acid polypeptides based upona heterocycle, including a nitrogen-containing heterocycle, linkage. Ina further embodiment are dicarbonyl-substituted molecules used toderivatize diamine-containing non-natural amino acid polypeptides viathe formation of a heterocycle, including a nitrogen-containingheterocycle group. In a further embodiment are dicarbonyl-substitutedmolecules that can form such heterocycle, including anitrogen-containing heterocycle groups with a diamine-containingnon-natural amino acid polypeptide in a pH range between about 4 andabout 8. In a further embodiment are dicarbonyl-substituted moleculesused to derivatize diamine-containing non-natural amino acidpolypeptides via the formation of a heterocycle, including anitrogen-containing heterocycle, linkage between the derivatizingmolecule and the diamine-containing non-natural amino acid polypeptides.In a further embodiment the dicarbonyl-substituted molecules arediketone-substituted molecules, in other aspectsketoaldehyde-substituted molecules, in other aspectsketoacid-substituted molecules, in other aspects ketoester-substitutedmolecules, including ketothioester-substituted molecules. In furtherembodiments, the dicarbonyl-substituted molecules comprise a groupselected from a desired functionality. In further or additionalembodiments, the aldehyde-substituted molecules are aldehyde-substitutedpolyethylene glycol (PEG) molecules. In a further embodiment, thesidechain of the non-natural amino acid has a chemistry orthogonal tothose of the naturally-occurring amino acids that allows the non-naturalamino acid to react selectively with the carbonyl-substituted molecules.In a further embodiment, the sidechain of the non-natural amino acidcomprises a moiety (e.g., diamine group) that reacts selectively withthe dicarbonyl-containing molecule; in a further embodiment, thenucleophilic moiety on the sidechain of the non-natural amino acid canundergo electrophilic attack to generate a heterocyclic-derivatizedprotein, including a nitrogen-containing heterocycle-derivatizedprotein. In a further aspect related to the embodiments described inthis paragraph are the modified non-natural amino acid polypeptides thatresult from the reaction of the derivatizing molecule with thenon-natural amino acid polypeptides. Further embodiments include anyfurther modifications of the already modified non-natural amino acidpolypeptides.

In one aspect are methods to derivatize proteins via the reaction ofcarbonyl and hydrazine reactants to generate a heterocycle-derivatizedprotein, including a nitrogen-containing heterocycle-derivatizeddolastatin. Included within this aspect are methods for thederivatization of dolastatin linker derivatives based upon thecondensation of carbonyl- and hydrazine-containing reactants to generatea heterocycle-derivatized dolastatin, including a nitrogen-containingheterocycle-derivatized dolastatin. In additional or further embodimentsare methods to derivatize ketone-containing dolastatin derivatives oraldehyde-containing dolastatin derivatives with hydrazine-functionalizednon-natural amino acids. In yet additional or further aspects, thehydrazine-substituted molecule can include proteins, other polymers, andsmall molecules.

In another aspect are methods for the chemical synthesis ofhydrazine-substituted molecules for the derivatization ofcarbonyl-substituted dolastatin derivatives. In one embodiment, thehydrazine-substituted molecule is a dolastatin linked derivativesuitable for the derivatization of carbonyl-containing non-natural aminoacid polypeptides, including by way of example only, ketone-, oraldehyde-containing non-natural amino acid polypeptides.

In one aspect are non-natural amino acids for the chemicalderivatization of dolastatin analogs based upon a quinoxaline orphenazine linkage. In further or additional embodiments, the non-naturalamino acids are functionalized on their sidechains such that theirreaction with a derivatizing dolastatin linker generates a quinoxalineor phenazine linkage. In further or additional embodiments, thenon-natural amino acids are selected from amino acids having1,2-dicarbonyl or 1,2-aryldiamine sidechains. In further or additionalembodiments, the non-natural amino acids are selected from amino acidshaving protected or masked 1,2-dicarbonyl or 1,2-aryldiaminesidechainis. Further included are equivalents to 1,2-dicarbonylsidechains, or protected or masked equivalents to 1,2-dicarbonylsidechains.

In another aspect are derivatizing molecules for the production ofderivatized non-natural amino acid polypeptides based upon quinoxalineor phenazine linkages. In one embodiment are 1,2-dicarbonyl substituteddolastatin linker derivatives used to derivatize 1,2-aryldiaminecontaining non-natural amino acid polypeptides to form quinoxaline orphenazine linkages. In another embodiment are 1,2-aryldiaminesubstituted dolastatin linker derivatives used to derivatize1,2-dicarbonyl containing non-natural amino acid polypeptides to formquinoxaline or phenazine linkages. In a further aspect related to theabove embodiments are the modified non-natural amino acid polypeptidesthat result from the reaction of the derivatizing dolastatin linker withthe non-natural amino acid polypeptides. In one embodiment are1,2-aryldiamine containing non-natural amino acid polypeptidesderivatized with 1,2-dicarbonyl substituted dolastatin linker derivativeto form quinoxaline or phenazine linkages. In another embodiment are1,2-dicarbonyl containing non-natural amino acid polypeptidesderivatized with 1,2-aryldiamine substituted dolastatin linkerderivatives to form quinoxaline or phenazine linkages.

Provided herein in certain embodiments are derivatizing molecules forthe production of toxic compounds comprising non-natural amino acidpolypeptides based upon triazole linkages. In some embodiments, thereaction between the first and second reactive groups can proceed via adipolarophile reaction. In certain embodiments, the first reactive groupcan be an azide and the second reactive group can be an alkyne. Infurther or alternative embodiments, the first reactive group can be analkyne and the second reactive group can be an azide. In someembodiments, the Huisgen cycloaddition reaction (see, e.g., Huisgen, in1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), p. 1-176)provides for the incorporation of non-naturally encoded amino acidsbearing azide and alkyne-containing side chains permits the resultantpolypeptides to be modified with extremely high selectivity. In certainembodiments, both the azide and the alkyne functional groups are inerttoward the twenty common amino acids found in naturally-occurringpolypeptides. When brought into close proximity, however, the“spring-loaded” nature of the azide and alkyne groups is revealed andthey react selectively and efficiently via Huisgen [3 2]cycloadditionreaction to generate the corresponding triazole. See, e.g., Chin et al.,Science 301:964-7 (2003); Wang et al., J. Am. Chem. Soc., 125, 3192-3193(2003); Chin et al., J. Am. Chem. Soc., 124:9026-9027 (2002).Cycloaddition reaction involving azide or alkyne-containing polypeptidescan be carried out at room temperature under aqueous conditions by theaddition of Cu(II) (e.g., in the form of a catalytic amount of CuSO₄) inthe presence of a reducing agent for reducing Cu(II) to Cu(I), in situ,in catalytic amount. See, e.g., Wang et al., J. Am. Chem. Soc. 125,3192-3193 (2003); Tornoe et al., J. Org. Chem. 67:3057-3064 (2002);Rostovtsev, Angew. Chem. Int. Ed. 41:2596-2599 (2002). Preferredreducing agents include ascorbate, metallic copper, quinine,hydroquinone, vitamin K, glutathione, cysteine, Fe², Co², and an appliedelectric potential.

Such non-natural amino acid heteroaryl-linked dolastatin derivativesinclude amino acids having the structure of Formula (XXXI), (XXXII),(XXXIII), (XXXIV), (XXXV), or (XXXVI):

wherein:

-   -   Z has the structure of:

-   -   -   R₅ is H, CO₂H, C₁-C₆alkyl, or thiazole;        -   R₆ is OH or H;        -   Ar is phenyl or pyridine;

    -   R₁ is H, an amino protecting group, resin, at least one amino        acid, polypeptide, or polynucleotide;

    -   R₂ is OH, an ester protecting group, resin, at least one amino        acid, polypeptide, or polynucleotide;

    -   R₄ is H, halogen, lower alkyl, or substituted lower alkyl;

    -   R₇ is C₁-C₆alkyl or hydrogen;

    -   L, L₁, L₂, L₃, and L₄ are each linkers selected from the group        consisting of a bond, -alkylene-, -alkylene-C(O)—, -alkylene-J-,        -(alkylene-O)_(n)-alkylene-, -(alkylene-O)_(n)-alkylene-C(O)—,        -(alkylene-O)_(n)-J-, -(alkylene-O)_(n)-J-alkylene-,        -(alkylene-O)_(n)—(CH₂)_(n′)—NHC(O)—CH₂)_(n″)—C(Me)₂-S—S—(CH₂)_(n′″)—NHC(O)-(alkylene-O)_(n″″)-alkylene-,        -(alkylene)-O)_(n)-alkylene-W—, -alkylene-C(O)—W—,        -(alkylene-O)_(n)-alkylene-J-,        -alkylene′-J-(alkylene-O)_(n)-alkylene-,        -(alkylene-O)_(n)-alkylene-J-alkylene′,        -J-(alkylene-O)_(n)-alkylene-,        -(alkylene-O)_(n)-alkylene-J-(alkylene-O)_(n)-alkylene-J′-, —W—,        -alkylene-W—, alkylene′-J-(alkylene-NMe)_(n)-alkylene-W—,        -J-(alkylene-NMe)_(n)-alkylene-W—,        -(alkylene-O)_(n)-alkylene-U-alkylene-C(O)—,        -(alkylene-O)_(n)-alkylene-U-alkylene-;        -J-alkylene-NMe-alkylene′-NMe-alkylene″-W—, and        -alkylene-J-alkylene′-NMe-alkylene″-NMe-alkylene′″-W—;

    -   W has the structure of:

-   -   -   U has the structure of:

-   -   -   each J and J′ independently have the structure of:

-   -   -   each n and n′ are independently integers greater than or            equal to one;

    -   D has the structure of:

-   -   -   each R₁₇ is independently selected from the group consisting            of H, alkyl, substituted alkyl, alkenyl, substituted            alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted            alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene            oxide, substituted polyalkylene oxide, aryl, substituted            aryl, heteroaryl, substituted heteroaryl, alkaryl,            substituted alkaryl, aralkyl, substituted aralkyl,            -(alkylene or substituted alkylene)-ON(R″)₂, -(alkylene or            substituted alkylene)-C(O)SR″, -(alkylene or substituted            alkylene)-S—S-(aryl or substituted aryl), —C(O)R″, —C(O)₂R″,            or —C(O)N(R″)₂, wherein each R″ is independently hydrogen,            alkyl, substituted alkyl, alkenyl, substituted alkenyl,            alkoxy, substituted alkoxy, aryl, substituted aryl,            heteroaryl, alkaryl, substituted alkaryl, aralkyl, or            substituted aralkyl;        -   each Z₁ is a bond, CR₁₇R₁₇, O, S, NR′, CR₁₇R₁₇—CR₁₇R₁₇,            CR₁₇R₁₇—O, O—CR₁₇R₁₇, CR₁₇R₁₇—S, S—CR₁₇R₁₇, CR₁₇R₁₇—NR′, or            NR′—CR₁₇R₁₇;            -   each R′ is H, alkyl, or substituted alkyl;        -   each Z₂ is selected from the group consisting of a bond,            —C(O)—, —C(S)—, optionally substituted C₁-C₃ alkylene,            optionally substituted C₁-C₃ alkenylene, and optionally            substituted heteroalkyl;        -   each Z₃ are independently selected from the group consisting            of a bond, optionally substituted C₁-C₄ alkylene, optionally            substituted C₁-C₄ alkenylene, optionally substituted            heteroalkyl, —O—, —S—, —C(O)—, —C(S)—, and —N(R′)—;        -   each T₃ is a bond, C(R″)(R″), O, or S; with the proviso that            when T is O or S, R″ cannot be halogen;        -   each R″ is H, halogen, alkyl, substituted alkyl, cycloalkyl,            or substituted cycloalkyl;        -   m and p are 0, 1, 2, or 3, provided that at least one of m            or p is not 0;        -   M₂ is

-   -    where (a) indicates bonding to the B group and (b) indicates        bonding to respective positions within the heterocycle group;        -   M₃ is

-   -    where (a) indicates bonding to the B group and (b) indicates        bonding to respective positions within the heterocycle group;        -   M₄ is

-   -    where (a) indicates bonding to the B group and (b) indicates        bonding to respective positions within the heterocycle group;        -   each R₁₉ is independently selected from the group consisting            of C₁-C₆ alkyl, C₁-C₆ alkoxy, ester, ether, thioether,            aminoalkyl, halogen, alkyl ester, aryl ester, amide, aryl            amide, alkyl halide, alkyl amine, alkyl sulfonic acid, alkyl            nitro, thioester, sulfonyl ester, halosulfonyl, nitrile,            alkyl nitrile, and nitro;        -   q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11; and    -   each R₁₆ is independently selected from the group consisting of        hydrogen, halogen, alkyl, NO₂, CN, and substituted alkyl.

In some embodiments, the compound of Formula (XXXI) include compoundshaving the structure of Formula (XXXI-A):

In certain embodiments of compounds of Formula (XXXI), (XXXII),(XXXIII), (XXXIV), (XXXV), or (XXXVI), R₅ is thiazole or carboxylicacid. In certain embodiments of compounds of Formula (XXXI), (XXXII),(XXIII), (XXXIV), (XXXV), or (XXXVI), R₆ is H. In certain embodiments ofcompounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or(XXXVI), Ar is phenyl. In certain embodiments of compounds of Formula(XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R₇ is methyl. Incertain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII),(XXXIV), (XXXV), or (XXXVI), n is an integer from 0 to 20. In certainembodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV),(XXXV), or (XXXVI), n is an integer from 0 to 10. In certain embodimentsof compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or(XXXVI), n is an integer from 0 to 5.

In certain embodiments of compounds of Formula (XXXI), (XXXII),(XXXIII), (XXXIV), (XXXV), or (XXXVI), R₅ is thiazole or carboxylicacid. In certain embodiments of compounds of Formula (XXXI), (XXXII),(XXXIII), (XXXIV), (XXXV), or (XXXVI), R₅ is hydrogen. In certainembodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV),(XXXV), or (XXXVI), R₅ is methyl, ethyl, propyl, iso-propyl, butyl,iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl. In certainembodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV),(XXXV), or (XXXVI), R₅ is —NH-(alkylene-O)_(n)—NH₂, wherein alkylene is—CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,or —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—. In certain embodiments ofFormula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), alkyleneis methylene, ethylene, propylene, butylenes, pentylene, hexylene, orheptylene.

In certain embodiments of compounds of Formula (XXXI), (XXXII),(XXXIII), (XXXIV), (XXXV), or (XXXVI), Rd₅ is —NH-(alkylene-O)—NH₂,wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments of compounds of Formula (XXXI), (XXXII),(XXXIII), (XXXIV), (XXXV), or (XXXVI), R₆ is H. In some embodiments ofcompounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or(XXXVI), R₆ is hydroxy.

In certain embodiments of compounds of Formula (XXXI), (XXXII),(XXXIII), (XXXIV), (XXXV), or (XXXVI), Ar is phenyl.

In certain embodiments of compounds of Formula (XXXI), (XXXII),(XXXIII), (XXXIV), (XXXV), or (XXXVI), R₇ is methyl, ethyl, propyl,iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. Incertain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII),(XXXIV), (XXXV), or (XXXVI), R₇ is hydrogen.

In certain embodiments of compounds of Formula (XXXI), (XXXII),(XXXIII), (XXXIV), (XXXV), or (XXXVI), each L, L₁, L₂, L₃, and L₄ isindependently a cleavable linker or non-cleavable linker. In certainembodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV),(XXXV), or (XXXVI), each L, L₁, L₂, L₃, and L₄ is independently aoligo(ethylene glycol) derivatized linker.

In certain embodiments of compounds of Formula (XXXI), (XXXII),(XXXIII), (XXXIV), (XXXV), or (XXXVI), each alkylene, alkylene′,alkylene″, and alkylene′″ independently is —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—,CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—, or—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—. In certain embodiments ofcompounds of Formula (XXXI), (XXXII), (XXXII), (XXXIV), (XXXV), or(XXXVI), alkylene is methylene, ethylene, propylene, butylenes,pentylene, hexylene, or heptylene.

In certain embodiments of compounds of Formula (XXXI), (XXXII),(XXXIII), (XXXIV), (XXXV), or (XXXVI), each n, n′, n″, n′″, and n″″independently is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments of compounds of Formula (XXXI), (XXXII),(XXXIII), (XXXIV), (XXXV), or (XXXVI), R₁ is a polypeptide. In certainembodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV),(XXXV), or (XXXVI), R₂ is a polypeptide. In certain embodiments ofcompounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or(XXXVI), the polypeptide is an antibody. In certain embodiments ofcompounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or(XXXVI), the antibody is αCD70.

Compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or(XXXVI) may be formed by the reductive alkylation of aromatic aminecompounds with carbonyl containing reagents such as, by way of example,ketones, esters, thioesters, and aldehydes.

The formation of such non-natural amino acid heterocycle-linkeddolastatin derivatives having the structure of Formula (XXXI), (XXXII),(XXXIII), (XXXIV), (XXXV), or (XXXVI) includes, but is not limited to,(i) reactions of diamine-containing non-natural amino acids withdicarbonyl-containing dolastatin linked derivatives or reactions ofdiamine-containing non-natural amino acids with ketoalkyne-containingdolastatin linked derivatives, (ii) reactions of dicarbonyl-containingnon-natural amino acids with either diamine-containing dolastatin linkedderivatives or reactions of dicarbonyl-containing non-natural aminoacids with ketoamine-containing dolastatin linked derivatives, (iii)reactions of ketoalkyne-containing non-natural amino acids withdiamine-containing dolastatin linked derivatives, or (iv) reactions ofketoamine-containing non-natural amino acids with dicarbonyl-containingv.

Modification of dolastatin linked derivatives described herein with suchreactions have any or all of the following advantages. First, diaminesundergo condensation with dicarbonyl-containing compounds in a pH rangeof about 5 to about 8 (and in further embodiments in a pH range of about4 to about 10, in other embodiments in a pH range of about 3 to about 8,in other embodiments in a pH range of about 4 to about 9, and in furtherembodiments a pH range of about 4 to about 9, in other embodiments a pHof about 4, and in yet another embodiment a pH of about 8) to generateheterocycle, including a nitrogen-containing heterocycle, linkages.Under these conditions, the sidechains of the naturally occurring aminoacids are unreactive. Second, such selective chemistry makes possiblethe site-specific derivatization of recombinant proteins: derivatizedproteins can now be prepared as defined homogeneous products. Third, themild conditions needed to effect the reaction of the diamines describedherein with the dicarbonyl-containing polypeptides described hereingenerally do not irreversibly destroy the tertiary structure of thepolypeptide (excepting, of course, where the purpose of the reaction isto destroy such tertiary structure). Fourth, the reaction occurs rapidlyat room temperature, which allows the use of many types of polypeptidesor reagents that would be unstable at higher temperatures. Fifth, thereaction occurs readily is aqueous conditions, again allowing use ofpolypeptides and reagents incompatible (to any extent) with non-aqueoussolutions. Six, the reaction occurs readily even when the ratio ofpolypeptide or amino acid to reagent is stoichiometric, nearstoichiometric, or stoichiometric-like, so that it is unnecessary to addexcess reagent or polypeptide to obtain a useful amount of reactionproduct. Seventh, the resulting heterocycle can be producedregioselectively and/or regiospecifically, depending upon the design ofthe diamine and dicarbonyl portions of the reactants. Finally, thecondensation of diamines with dicarbonyl-containing molecules generatesheterocycle, including a nitrogen-containing heterocycle, inkages whichare stable under biological conditions.

VI. Location of Non-Natural Amino Acids in Dolastatin Linker Derivatives

The methods and compositions described herein include incorporation ofone or more non-natural amino acids into a dolastatin linker derivative.One or more non-natural amino acids may be incorporated at one or moreparticular positions which do not disrupt activity of the dolastatinlinker derivative. This can be achieved by making “conservative”substitutions, including but not limited to, substituting hydrophobicamino acids with non-natural or natural hydrophobic amino acids, bulkyamino acids with non-natural or natural bulky amino acids, hydrophilicamino acids with non-natural or natural hydrophilic amino acids) and/orinserting the non-natural amino acid in a location that is not requiredfor activity.

A variety of biochemical and structural approaches can be employed toselect the desired sites for substitution with a non-natural amino acidwithin the dolastatin linker derivative. In some embodiments, thenon-natural amino acid is linked at the C-terminus of the dolastatinderivative. In other embodiments, the non-natural amino acid is linkedat the N-terminus of the dolastatin derivative. Any position of thedolastatin linker derivative is suitable for selection to incorporate anon-natural amino acid, and selection may be based on rational design orby random selection for any or no particular desired purpose. Selectionof desired sites may be based on producing a non-natural amino acidpolypeptide (which may be further modified or remain unmodified) havingany desired property or activity, including but not limited to areceptor binding modulators, receptor activity modulators, modulators ofbinding to binder partners, binding partner activity modulators, bindingpartner conformation modulators, dimer or multimer formation, no changeto activity or property compared to the native molecule, or manipulatingany physical or chemical property of the polypeptide such as solubility,aggregation, or stability. Alternatively, the sites identified ascritical to biological activity may also be good candidates forsubstitution with a non-natural amino acid, again depending on thedesired activity sought for the polypeptide. Another alternative wouldbe to simply make serial substitutions in each position on thepolypeptide chain with a non-natural amino acid and observe the effecton the activities of the polypeptide. Any means, technique, or methodfor selecting a position for substitution with a non-natural amino acidinto any polypeptide is suitable for use in the methods, techniques andcompositions described herein.

The structure and activity of naturally-occurring mutants of apolypeptide that contain deletions can also be examined to determineregions of the protein that are likely to be tolerant of substitutionwith a non-natural amino acid. Once residues that are likely to beintolerant to substitution with non-natural amino acids have beeneliminated, the impact of proposed substitutions at each of theremaining positions can be examined using methods including, but notlimited to, the three-dimensional structure of the relevant polypeptide,and any associated ligands or binding proteins. X-ray crystallographicand NMR structures of many polypeptides are available in the ProteinData Bank (PDB, www.rcsb.org), a centralized database containingthree-dimensional structural data of large molecules of proteins andnucleic acids, one can be used to identify amino acid positions that canbe substituted with non-natural amino acids. In addition, models may bemade investigating the secondary and tertiary structure of polypeptides,if three-dimensional structural data is not available. Thus, theidentity of amino acid positions that can be substituted withnon-natural amino acids can be readily obtained.

Exemplary sites of incorporation of a non-natural amino acid include,but are not limited to, those that are excluded from potential receptorbinding regions, or regions for binding to binding proteins or ligandsmay be fully or partially solvent exposed, have minimal or nohydrogen-bonding interactions with nearby residues, may be minimallyexposed to nearby reactive residues, and/or may be in regions that arehighly flexible as predicted by the three-dimensional crystal structureof a particular polypeptide with its associated receptor, ligand orbinding proteins.

A wide variety of non-natural amino acids can be substituted for, orincorporated into, a given position in a polypeptide. By way of example,a particular non-natural amino acid may be selected for incorporationbased on an examination of the three dimensional crystal structure of apolypeptide with its associated ligand, receptor and/or bindingproteins, a preference for conservative substitutions

In one embodiment, the methods described herein include incorporatinginto the dolastatin linker derivative, where the dolastatin linkerderivative comprises a first reactive group; and contacting thedolastatin linker derivative with a molecule (including but not limitedto a second protein or polypeptide or polypeptide analog; an antibody orantibody fragment; and any combination thereof) that comprises a secondreactive group. In certain embodiments, the first reactive group is ahydroxylamine moiety and the second reactive group is a carbonyl ordicarbonyl moiety, whereby an oxime linkage is formed. In certainembodiments, the first reactive group is a carbonyl or dicarbonyl moietyand the second reactive group is a hydroxylamine moiety, whereby anoxime linkage is formed. In certain embodiments, the first reactivegroup is a carbonyl or dicarbonyl moiety and the second reactive groupis an oxime moiety, whereby an oxime exchange reaction occurs. Incertain embodiments, the first reactive group is an oxime moiety and thesecond reactive group is carbonyl or dicarbonyl moiety, whereby an oximeexchange reaction occurs.

In some cases, the dolastatin linker derivative incorporation(s) will becombined with other additions, substitutions, or deletions within thepolypeptide to affect other chemical, physical, pharmacologic and/orbiological traits. In some cases, the other additions, substitutions ordeletions may increase the stability (including but not limited to,resistance to proteolytic degradation) of the polypeptide or increaseaffinity of the polypeptide for its appropriate receptor, ligand and/orbinding proteins. In some cases, the other additions, substitutions ordeletions may increase the solubility (including but not limited to,when expressed in E. coli or other host cells) of the polypeptide. Insome embodiments sites are selected for substitution with a naturallyencoded or non-natural amino acid in addition to another site forincorporation of a non-natural amino acid for the purpose of increasingthe polypeptide solubility following expression in E. coli, or otherrecombinant host cells. In some embodiments, the polypeptides compriseanother addition, substitution, or deletion that modulates affinity forthe associated ligand, binding proteins, and/or receptor, modulates(including but not limited to, increases or decreases) receptordimerization, stabilizes receptor dimers, modulates circulatinghalf-life, modulates release or bio-availability, facilitatespurification, or improves or alters a particular route ofadministration. Similarly, the non-natural amino acid polypeptide cancomprise chemical or enzyme cleavage sequences, protease cleavagesequences, reactive groups, antibody-binding domains (including but notlimited to, FLAG or poly-His) or other affinity based sequences(including but not limited to, FLAG, poly-His, GST, etc.) or linkedmolecules (including but not limited to, biotin) that improve detection(including but not limited to, GFP), purification, transport thrutissues or cell membranes, prodrug release or activation, sizereduction, or other traits of the polypeptide.

VII. Anti-CD70 Expression and Ligand Interaction as Exemplar

The methods, compositions, strategies and techniques described hereinare not limited to a particular type, class or family of polypeptides orproteins. Indeed, virtually any polypeptides may be designed or modifiedto include at least one “modified or unmodified” non-natural amino acidscontaining dolastatin linker derivative described herein. By way ofexample only, the polypeptide can be homologous to a therapeutic proteinselected from the group consisting of: alpha-1 antitrypsin, angiostatin,antihemolytic factor, antibody, antibody fragment, monoclonal antibody(e.g., bevacizumab, cetuximab, panitumumab, infliximab, adalimumab,basiliximab, daclizumab, omalizumab, ustekinumab, etanercept,gemtuzumab, alemtuzumab, rituximab, trastuzumab, nimotuzumab,palivizumab, and abciximnab), apolipoprotein, apoprotein, atrialnatriuretic factor, atrial natriuretic polypeptide, atrial peptide,C—X—C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10,GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit ligand, cytokine, CCchemokine, monocyte chemoattractant protein-1, monocyte chemoattractantprotein-2, monocyte chemoattractant protein-3, monocyte inflammatoryprotein-1 alpha, monocyte inflammatory protein-i beta, RANTES, 1309,R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kitligand, collagen, colony stimulating factor (CSF), complement factor 5a,complement inhibitor, complement receptor 1, cytokine, epithelialneutrophil activating peptide-78, MIP-16, MCP-1, epidermal growth factor(EGF), epithelial neutrophil activating peptide, erythropoietin (EPO),exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X,fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helicalbundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin,growth factor, growth factor receptor, grf, hedgehog protein,hemoglobin, hepatocyte growth factor (hGF), hirudin, human growthhormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1,LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I,IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemiainhibitory factor, luciferase, neurturin, neutrophil inhibitory factor(NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin,parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin,protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B,pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosyntheticprotein, soluble complement receptor I, soluble I-CAM 1, solubleinterleukin receptor, soluble TNF receptor, somatomedin, somatostatin,somatotropin, streptokinase, superantigens, staphylococcal enterotoxin,SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor,superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1,tissue plasminogen activator, tumor growth factor (TOF), tumor necrosisfactor, tumor necrosis factor alpha, tumor necrosis factor beta, tumornecrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascularendothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53,tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor,testosterone receptor, aldosterone receptor, LDL receptor, andcorticosterone.

Thus, the following description of an ARX-anti-CD70 ADC,anti-CD70-2H5-HA119-NCA, and CD70-2H5-HA119-NCA1 are provided forillustrative purposes and by way of non-limiting example of the scope ofthe methods, compositions, strategies and techniques described herein.

CD70-2H5-HA119-NCA1 is a humanized monoclonal antibody drug conjugatethat interacts with the CD27 ligand which functions, by non-limitingexample, by promoting cell survival and expansion of antigen primed CD8T cells, formation of memory T cells and proliferation of B cells. CD70receptors have been found in high densities in cancer tissue, forexample 34000-189000 copies per cell (Caki-1, 786-O, L-428, UMRC3, LP-1,DBTRG-05 MG) (Engineered anti-CD70 antibody-drug conjugate withincreased therapeutic index, Molecular Cancer Therapeutics, 2008) and innon-cancerous tissue and/or normal tissue the receptors are on 5%-15% ofactivated T cells and 10%-25% of activated B cells. CD70 expression hasbeen found on ˜40% of multiple myeloma isolates (PreclinicalCharacterization of SGN-70, a Humanized Antibody Directed against CD70,Cancer Therapy: Preclinical, 2008) and confirmed CD70 expression on ahigh percentage of, for example, Hodgkin lymphoma Reed-Steinberg cells,non-Hodgkin lymphoma, and renal cell carcinoma tumors. CD70 is a type IIintegral membrane protein of the TNF family. For purposes of thisinvention, it is possible for the αCD70 antibody to be any known CD70antibody with one non-naturally encoded amino acid. For purposes ofillustration, CD70-2H5-HA119-NCA1 antibodies are described in Table 1.

In one embodiment of the present invention, the antibody comprises ananti-CD70 constant region comprising SEQ ID NO: 3 with one non-naturallyencoded amino acid. In another embodiment of the present invention, theantibody comprises an anti-CD70 constant region comprising SEQ ID NO: 4.In one embodiment of the present invention, the antibody comprises ananti-CD70 constant region comprising SEQ ID NO: 3 where one amino acidis a non-naturally encoded amino acid. In one embodiment of the presentinvention, the antibody comprises an anti-CD70 constant regioncomprising SEQ ID NO: 3 where the first amino acid is a non-naturallyencoded amino acid. In one embodiment of the present invention, theantibody comprises an anti-CD70 constant region comprising SEQ ID NO: 4where one amino acid is a non-naturally encoded amino acid. In oneembodiment of the present invention, the antibody comprises an anti-CD70constant region comprising SEQ ID NO: 4 where the first amino acid is anon-naturally encoded amino acid. In one embodiment of the presentinvention, the antibody comprises the αCD70 antibody with a variableheavy chain of SEQ ID NO: 1. In another embodiment of the presentinvention, the antibody comprises the αCD70 antibody with a variableheavy chain of SEQ ID NO: 10. In one embodiment of the presentinvention, the antibody comprises the αCD70 antibody with a variablelight chain of SEQ ID NO: 2. In another embodiment of the presentinvention, the antibody comprises the αCD70 antibody with a variablelight chain of SEQ ID NO: 11. In another embodiment of the presentinvention, the antibody comprises a heavy chain of SEQ ID NO. 1 and alight chain of SEQ ID NO. 2. In another embodiment of the presentinvention, the antibody comprises a heavy chain of SEQ ID NO. 10 and alight chain of SEQ ID NO. 11. In another embodiment of the presentinvention, the antibody comprises a heavy chain of SEQ ID NO. 1 and alight chain of SEQ ID NO. 2 and a constant region. In another embodimentof the present invention, the antibody comprises a heavy chain of SEQ IDNO. 10 and a light chain of SEQ ID NO. 11 and a constant region. Inanother embodiment of the present invention, the antibody comprises aheavy chain of SEQ ID NO. 10 and a light chain of SEQ ID NO. 11 and aconstant region comprising SEQ ID NO.3. In another embodiment of thepresent invention, there is one non-naturally encoded amino acid in theanti-CD70 antibody sequence. In another embodiment of the presentinvention, there is more than one non-naturally encoded amino acid inthe anti-CD70 antibody sequence. In another embodiment of the presentinvention, amino acid 119 of SEQ ID NO. 1 is substituted with anon-naturally encoded amino acid. In another embodiment of the presentinvention, amino acid 122 of SEQ ID NO. 10 is substituted with anon-naturally encoded amino acid. In another embodiment of the presentinvention, the antibody comprises a heavy chain of SEQ ID NO. 1 and alight chain of SEQ ID NO. 2 and a constant region comprising SEQ ID NO.3. In other embodiments of the present invention, more than onenon-naturally encoded amino acid is substituted into the anti-CD70antibody. In other embodiments of the present invention, the αCD70antibody comprises a heavy chain with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10non-naturally encoded amino acids. In other embodiments of the presentinvention, the αCD70 antibody comprises SEQ ID NO. 4 and anti-CD70variable regions provided in U.S. Published Patent Application20100150950.

In other embodiments of the present invention, the antibody is an αCD70antibody comprising an amino acid sequence with one or morenon-naturally encoded amino acids and 90% homology to SEQ ID NO: 1. Inother embodiments of the present invention, the antibody is an αCD70antibody comprising an amino acid sequence with one or morenon-naturally encoded amino acids and 90% homology to SEQ ID NO: 2. Inother embodiments of the present invention, the antibody is an αCD70antibody comprising an amino acid sequence with one or morenon-naturally encoded amino acids and 90% homology to SEQ ID NO: 3. Inother embodiments of the present invention, the antibody is an αCD70antibody comprising an amino acid sequence with one or morenon-naturally encoded amino acids and 95% homology to SEQ ID NO: 1. Inother embodiments of the present invention, the antibody is an αCD70antibody comprising an amino acid sequence with one or morenon-naturally encoded amino acids and 95% homology to SEQ ID NO: 2. Inother embodiments of the present invention, the antibody is an αCD70antibody comprising an amino acid sequence with one or morenon-naturally encoded amino acids and 95% homology to SEQ ID NO: 3. Inother embodiments of the present invention, the antibody is an αCD70antibody comprising an amino acid sequence with one or morenon-naturally encoded amino acids and 96% homology to SEQ ID NO: 1. Inother embodiments of the present invention, the antibody is an αCD70antibody comprising an amino acid sequence with one or morenon-naturally encoded amino acids and 96% homology to SEQ ID NO: 2. Inother embodiments of the present invention, the antibody is an αCD70antibody comprising an amino acid sequence with one or morenon-naturally encoded amino acids and 96% homology to SEQ ID NO: 3. Inother embodiments of the present invention, the antibody is an αCD70antibody comprising an amino acid sequence with one or morenon-naturally encoded amino acids and 97% homology to SEQ ID NO: 1. Inother embodiments of the present invention, the antibody is an αCD70antibody comprising an amino acid sequence with one or morenon-naturally encoded amino acids and 97% homology to SEQ ID NO: 2. Inother embodiments of the present invention, the antibody is an αCD70antibody comprising an amino acid sequence with one or morenon-naturally encoded amino acids and 97% homology to SEQ ID NO: 3. Inother embodiments of the present invention, the antibody is an αCD70antibody comprising an amino acid sequence with one or morenon-naturally encoded amino acids and 98% homology to SEQ ID NO: 1. Inother embodiments of the present invention, the antibody is an αCD70antibody comprising an amino acid sequence with one or morenon-naturally encoded amino acids and 98% homology to SEQ ID NO: 2. Inother embodiments of the present invention, the antibody is an αCD70antibody comprising an amino acid sequence with one or morenon-naturally encoded amino acids and 98% homology to SEQ ID NO: 3. Inother embodiments of the present invention, the antibody is an αCD70antibody comprising an amino acid sequence a non-naturally encoded aminoacids and 99% homology to SEQ ID NO: 1. In other embodiments of thepresent invention, the antibody is an αCD70 antibody comprising an aminoacid sequence a non-naturally encoded amino acids and 99% homology toSEQ ID NO: 2. In other embodiments of the present invention, theantibody is an αCD70 antibody comprising an amino acid sequence anon-naturally encoded amino acids and 99% homology to SEQ ID NO: 3.Non-naturally encoded amino acid site selection is described within thisspecification, and as it specifically pertains to α-CD70 antibodies,sites were selected based on surface exposure/site accessibility withinthe antibody and hydrophobic/neutral amino acid sites were selected tomaintain the charge on the antibody.

TABLE 1 ANTIBODY AMINO ACID AND NUCLEOTIDE SEQUENCES SEQ ID NO.IDENTIFIER SEQUENCE  1 Humanized 7F2EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMNWVQQA VH amino acidPGKGLEWMGIINPYNGGTHYNQKFKGRVTITADTSTDTAYMELSSLRSEDTAVYYCATSGYDLYFDYWGQGTLVTVSS* (Amber substitution at A119)  2Humanized 7F2 EIVMTQSPATLSVSPGERATLSCKASQNVGTAVAWYQQKPG VL amino acidQAPRLLIYSAFNRYNGIPARFSGSGPGTDFTLTISSLQSED FAVYYCQQYSTYPLTFGGGTKVEIK  3Constant STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS Region, GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN 234-236HKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGGPSVFLFPP mutations KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN amino acidAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*  4 ConstantSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS Region,GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN Wild Type  HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK amino acidPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG  5 Humanized 7F2RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK Kappa aminoVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV acid YACEVTHQGLSSPVTKSFNRGEC* 6 Humanized 7F2 GAGGTGCAGCTGGTTCAGTCTGGGGCCGAGGTGAAAAAGC VH nucleotideCTGGGGCCACCGTGAAAATTTCTTGTAAAGTTTCTGGATATACCTTCACCGATTACTATATGAACTGGGTTCAGCAAGCCCCTGGAAAGGGACTGGAGTGGATGGGGATAATTAACCCATATAACGGCGGCACCCACTATAACCAAAAGTTCAAAGGGAGGGTTACCATCACTGCAGACACCAGCACCGACACCGCTTATATGGAGCTGAGTAGCCTGAGGTCCGAGGATACTGCAGTTTACTATTGTGCGACTTCCGGCTACGATCTGTACTTTGACTACTGGGGCCAGGGAACCCTCGTTACCGTCTCCTCA  7 Humanized 7F2GAAATTGTGATGACACAGTCTCCGGCCACCCTGTCTGTTTC VL nucleotideTCCGGGGGAAAGAGCTACCCTGAGTTGCAAAGCCAGTCAGAACGTTGGGACCGCCGTTGCCTGGTACCAACAGAAACCTGGCCAGGCCCCCCGTCTCCTCATCTATTCCGCATTCAACAGGTATAACGGCATCCCAGCAAGGTTCTCCGGCAGTGGGCCAGGGACAGACTTCACTCTCACCATCAGCAGCCTGCAGAGCGAGGACTTCGCAGTTTATTATTGTCAGCAGTACAGTACCTACCCTCTCACTTTCGGAGGAGGCACTAAAGTTGAGATCAAA  8 7F2 ConstantGCACCTCCCGTCGCCGGGGGACCGTCAGTCTTCCTCTTCCC Region, 234-236CCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCT mutationsGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACC nucleotideCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCT GTCTCCGGGTTGA  9 Humanized 7F2CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATC KappaTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCC nucleotideTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT TGA 10 Humanized 8A1QVQLVQSGAEVKKPGSSVKVSCKASGYSFSDYYMNWVRQA VH amino acidPGQGLEWMGEINPTTGGATYNQKFKARVTITADESTSTAYMELSSLRSEDTAVYYCARSLFPANYAGFVYWGQGTLVTVSS* (Amber substitution at A122)11 Humanized 8A1 DIQMTQSPSSLSASVGDRVTITCSASSSVSYMYWYQQKPGKAVL amino acid PKLLIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSYPWTFGQGTKVEIK 12 Humanized 8A1RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK Kappa aminoVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV acid YACEVTHQGLSSPVTKSFNRGEC*13 Humanized 8A1 CAGGTGCAGCTGGTTCAGTCTGGGGCCGAGGTGAAAAAGC VH nucleotideCTGGGTCTTCTGTGAAAGTTTCTTGTAAAGCAAGTGGATATTCTTTCAGTGATTACTATATGAACTGGGTTAGGCAGGCCCCTGGCCAAGGACTCGAGTGGATGGGGGAGATTAACCCAACCACCGGCGGGGCAACCTATAACCAAAAGTTCAAAGCAAGGGTTACCATCACTGCTGACGAGAGCACTTCAACTGCTTATATGGAGCTGAGCAGCCTGAGGTCCGAGGATACCGCAGTTTACTATTGTGCGAGATCCCTCTTCCCTGCAAACTACGCAGGCTTCGTTTACTGGGGACAGGGAACCCTGGTCACCGTCTCCTCA 14 Humanized 8A1GATATTCAGATGACACAGTCTCCGTCTTCTCTGTCTGCCAG VL nucleotideTGTTGGGGATAGAGTTACCATTACCTGCAGTGCCAGTTCTAGTGTTTCCTACATGTACTGGTACCAACAGAAACCTGGCAAGGCCCCCAAACTCCTCATCTATGACACCTCCAAACTCGCAAGTGGCGTTCCATCCAGGTTCTCCGGCAGTGGGAGTGGGACAGACTATACTTTCACCATCAGCAGCCTCCAGCCTGAGGACATCGCAACCTATTATTGTCAGCAGTGGAGTAGCTACCCTTGGACTTTCGGACAGGGAACGAAAGTTGAGATCAAACGTAC GGTGGCTGCACCATCTGT 158A1 Constant TAGAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT Region, 234-236CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTG mutationsCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG nucleotideTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTCGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC AGAAGAGCCTCTCCCTGTCTCCGGGTTGA16 Humanized 8A1 CTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAA KappaCTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGA nucleotideGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGC TTCAACAGGGGAGAGTGTTGA 17ARX-anti-CD70 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYIMHWVRQAP 2H5 VHGKGLEWVAVISYDGRNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDTDGYDFDYWGQGTLVTVSS 18 ARX-anti-CD70EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQ 2H5 VLAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFA VYYCQQRTNWPLTFGGGTKVEIK

In one embodiment, the present invention provides an αCD70 antibody drugconjugate (ADC or an αCD70-ADC) wherein the antibody is an αCD70comprising a light and heavy chain. In another embodiment of the presentinvention is provided an αCD70-ADC comprising a heavy chain (SEQ. ID.NO.: 1). In another embodiment of the present invention is provided anαCD70-ADC comprising a light (SEQ. ID. NO.: 2). In another embodiment ofthe present invention is provided an αCD70-ADC comprising a heavy chain(SEQ. ID. NO.: 10). In another embodiment of the present invention isprovided an αCD70-ADC comprising a light (SEQ. ID. NO.: 11). In anotherembodiment of the present invention is provided an αCD70-ADC comprisinga light (SEQ. ID. NO.: 2) and heavy chain (SEQ. ID. NO.: 1). In anotherembodiment of the present invention is provided an αCD70-ADC comprisinga light (SEQ. ID. NO.: 11) and heavy chain (SEQ. ID. NO.: 10). In someembodiments of the present invention the constant region, SEQ ID NO. 3or 4, has mutated any of the amino acid positions selected from thegroup 115, 116, 117. In some embodiments of the present invention,mutations are made to the antibody to inhibit ADCC function. In someembodiments of the present invention, the antibody comprises an αCD70antibody with one or more non-naturally encoded amino acids. In anotherembodiment of the present invention, the antibody comprises an αCD70antibody with one or more non-naturally encoded amino acids substitutedat one or more positions of SEQ ID NO: 1. In another embodiment of thepresent invention, the antibody comprises an αCD70 antibody with one ormore non-naturally encoded amino acids substituted at 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 positions of SEQ ID NO: 1. In another embodiment of thepresent invention, the antibody comprises an αCD70 antibody with onenon-naturally encoded amino acid substituted at a position in SEQ IDNO: 1. In another embodiment of the present invention, the antibodycomprises an αCD70 antibody with one non-naturally encoded amino acidssubstituted at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of SEQ IDNO: 1. In another embodiment of the present invention, the antibodycomprises an αCD70 antibody with one or more non-naturally encoded aminoacids substituted at one or more positions of SEQ ID NO: 2. In anotherembodiment of the present invention, the antibody comprises an αCD70antibody with one or more non-naturally encoded amino acids substitutedat 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of SEQ ID NO: 2. Inanother embodiment of the present invention, the antibody comprises anαCD70 antibody with one non-naturally encoded amino acid substituted ata position in SEQ ID NO: 2. In another embodiment of the presentinvention, the antibody comprises an αCD70 antibody with onenon-naturally encoded amino acids substituted at 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 positions of SEQ ID NO: 2.

In another embodiment of the present invention, the antibody comprisesan αCD70 antibody with one or more non-naturally encoded amino acidssubstituted at one or more positions of SEQ ID NO: 3. In anotherembodiment of the present invention, the antibody comprises an αCD70antibody with one or more non-naturally encoded amino acids substitutedat 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of SEQ ID NO: 3. Inanother embodiment of the present invention, the antibody comprises anαCD70 antibody with one non-naturally encoded amino acid substituted ata position in SEQ ID NO: 3. In another embodiment of the presentinvention, the antibody comprises an αCD70 antibody with onenon-naturally encoded amino acids substituted at 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 positions of SEQ ID NO: 3.

In another embodiment of the present invention, the antibody comprisesan αCD70 antibody with one or more non-naturally encoded amino acidssubstituted at one or more positions of SEQ ID NO: 10. In anotherembodiment of the present invention, the antibody comprises an αCD70antibody with one or more non-naturally encoded amino acids substitutedat 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of SEQ ID NO: 10. Inanother embodiment of the present invention, the antibody comprises anαCD70 antibody with one non-naturally encoded amino acid substituted ata position in SEQ ID NO: 10. In another embodiment of the presentinvention, the antibody comprises an αCD70 antibody with onenon-naturally encoded amino acids substituted at 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 positions of SEQ ID NO: 10.

In another embodiment of the present invention, the antibody comprisesan αCD70 antibody with one or more non-naturally encoded amino acidssubstituted at one or more positions of SEQ ID NO: 11. In anotherembodiment of the present invention, the antibody comprises an αCD70antibody with one or more non-naturally encoded amino acids substitutedat 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of SEQ ID NO: 11. Inanother embodiment of the present invention, the antibody comprises anαCD70 antibody with one non-naturally encoded amino acid substituted ata position in SEQ ID NO: 11. In another embodiment of the presentinvention, the antibody comprises an αCD70 antibody with onenon-naturally encoded amino acids substituted at 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 positions of SEQ ID NO: 11.

In another embodiment of the present invention, the antibody comprisesan αCD70 antibody with one or more non-naturally encoded amino acidssubstituted at one or more positions of SEQ ID NO: 4. In anotherembodiment of the present invention, the antibody comprises an αCD70antibody with one or more non-naturally encoded amino acids substitutedat 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of SEQ ID NO: 4. Inanother embodiment of the present invention, the antibody comprises anαCD70 antibody with one non-naturally encoded amino acid substituted ata position in SEQ ID NO: 4. In another embodiment of the presentinvention, the antibody comprises an αCD70 antibody with onenon-naturally encoded amino acids substituted at 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 positions of SEQ ID NO: 4.

In another embodiment of the present invention, the antibody comprisesan αCD70 antibody with one or more non-naturally encoded amino acidssubstituted at 1, 2, 3, 4, or 5 positions of SEQ ID NO: 1. In anotherembodiment of the present invention, the antibody comprises an αCD70antibody with one or more non-naturally encoded amino acids substitutedat 1, 2, or 3 positions of SEQ ID NO: 1. In another embodiment of thepresent invention, the antibody comprises an αCD70 antibody with one ormore non-naturally encoded amino acids substituted at 1 or 2 positionsof SEQ ID NO: 1. In another embodiment of the present invention, theantibody comprises an αCD70 antibody with two non-naturally encodedamino acids substituted at 2 positions of SEQ ID NO: 1. In anotherembodiment of the present invention, the antibody comprises an αCD70antibody with one non-naturally encoded amino acids substituted at onepositions of SEQ ID NO: 1. In another embodiment of the presentinvention, the antibody comprises the αCD70 antibody of SEQ ID NO: 3. Inanother embodiment of the present invention, the antibody comprises theαCD70 antibody of SEQ ID NO: 4. In another embodiment of the presentinvention, the antibody comprises the αCD70 antibody of SEQ ID NO: 5. Inanother embodiment of the present invention, the antibody comprises theαCD70 antibody of SEQ ID NO: 6. In another embodiment of the presentinvention, the antibody comprises the αCD70 antibody of SEQ ID NO: 7. Inanother embodiment of the present invention, the antibody comprises theαCD70 antibody of SEQ ID NO: 8. In another embodiment of the presentinvention, the antibody comprises the αCD70 antibody of SEQ ID NO: 1 andan αCD70 antibody heavy chain. In another embodiment of the presentinvention, the antibody comprises the αCD70 antibody of SEQ ID NO: 1 andan αCD70 antibody heavy chain of SEQ ID NO. 2 wherein the heavy chaincomprises one or more non-naturally encoded amino acids. In anotherembodiment of the present invention, the antibody comprises the αCD70antibody of SEQ ID NO: 1 and an αCD70 antibody heavy chain of SEQ ID NO.2 wherein the heavy chain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10non-naturally encoded amino acids. In another embodiment of the presentinvention, the antibody comprises the αCD70 antibody of SEQ ID NO: 1 andan αCD70 antibody heavy chain of SEQ ID NO. 2 wherein the heavy chaincomprises 1, 2, 3, 4, or 5 non-naturally encoded amino acids. In anotherembodiment of the present invention, the antibody comprises the αCD70antibody of SEQ ID NO: 1 and an αCD70 antibody heavy chain of SEQ ID NO.2 wherein the heavy chain comprises 1, 2, or 3 non-naturally encodedamino acids. In another embodiment of the present invention, theantibody comprises the αCD70 antibody of SEQ ID NO: 1 and an αCD70antibody heavy chain of SEQ ID NO. 2 wherein the heavy chain comprisestwo non-naturally encoded amino acids. In another embodiment of thepresent invention, the antibody comprises the αCD70 antibody of SEQ IDNO: 1 and an αCD70 antibody heavy chain of SEQ ID NO. 2 wherein theheavy chain comprises one non-naturally encoded amino acid. In anotherembodiment, the present invention provides an αCD70-ADC comprising SEQID NO: 1 and SEQ ID NO: 9. In another embodiment, the present inventionprovides an αCD70-ADC comprising SEQ ID NO: 1 and SEQ ID NO: 10. Inanother embodiment, the present invention provides an αCD70-ADCcomprising SEQ ID NO: 1 and SEQ ID NO: 11. In another embodiment, thepresent invention provides an αCD70-ADC comprising SEQ ID NO: 1 and SEQID NO: 12. In another embodiment, the present invention provides anαCD70-ADC comprising SEQ ID NO: 1 and SEQ ID NO: 13. In anotherembodiment, the present invention provides an αCD70-ADC comprising SEQID NO: 1 and SEQ ID NO: 14.

In another embodiment of the present invention, the antibody comprisesan αCD70 antibody with one or more non-naturally encoded amino acidssubstituted at one or more positions of SEQ ID NO: 2. In anotherembodiment of the present invention, the antibody comprises an αCD70antibody with one or more non-naturally encoded amino acids substitutedat 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of SEQ ID NO: 2. Inanother embodiment of the present invention, the antibody comprises anαCD70 antibody with one or more non-naturally encoded amino acidssubstituted at 1, 2, 3, 4, or 5 positions of SEQ ID NO: 2. In anotherembodiment of the present invention, the antibody comprises an αCD70antibody with one or more non-naturally encoded amino acids substitutedat 1, 2, or 3 positions of SEQ ID NO: 2. In another embodiment of thepresent invention, the antibody comprises an αCD70 antibody with one ormore non-naturally encoded amino acids substituted at 1 or 2 positionsof SEQ ID NO: 2. In another embodiment of the present invention, theantibody comprises an αCD70 antibody with two non-naturally encodedamino acids substituted at 2 positions of SEQ ID NO: 2. In anotherembodiment of the present invention, the antibody comprises an αCD70antibody with one non-naturally encoded amino acids substituted at onepositions of SEQ ID NO: 2. In another embodiment of the presentinvention, the antibody comprises the αCD70 antibody of SEQ ID NO: 9. Inanother embodiment of the present invention, the antibody comprises theαCD70 antibody of SEQ ID NO: 10. In another embodiment of the presentinvention, the antibody comprises the αCD70 antibody of SEQ ID NO: 11.In another embodiment of the present invention, the antibody comprisesthe αCD70 antibody of SEQ ID NO: 12. In another embodiment of thepresent invention, the antibody comprises the αCD70 antibody of SEQ IDNO: 13. In another embodiment of the present invention, the antibodycomprises the αCD70 antibody of SEQ ID NO: 14. In another embodiment ofthe present invention, the antibody comprises the αCD70 antibody of SEQID NO: 2 and an αCD70 antibody light chain. In another embodiment of thepresent invention, the antibody comprises the αCD70 antibody of SEQ IDNO: 9 and an αCD70 antibody light chain. In another embodiment of thepresent invention, the antibody comprises the αCD70 antibody of SEQ IDNO: 10 and an αCD70 antibody light chain. In another embodiment of thepresent invention, the antibody comprises the αCD70 antibody of SEQ IDNO: 11 and an αCD70 antibody light chain. In another embodiment of thepresent invention, the antibody comprises the αCD70 antibody of SEQ IDNO: 12 and an αCD70 antibody light chain. In another embodiment of thepresent invention, the antibody comprises the αCD70 antibody of SEQ IDNO: 13 and an αCD70 antibody light chain. In another embodiment of thepresent invention, the antibody comprises the αCD70 antibody of SEQ IDNO: 14 and an αCD70 antibody light chain.

As an additional, non-limiting example, the following description ofHER2 antibody trastuzumab is provided for illustrative purposes and byway of example only, and not as a limit on the scope of the methods,compositions, strategies and techniques described herein. Further,reference to trastuzumab in this application is intended to use thegeneric term as an example of any antibody. Thus, it is understood thatthe modifications and chemistries described herein with reference totrastuzumab can be equally applied to any antibody or monoclonalantibody, including those specifically listed herein.

Trastuzumab is a humanized monoclonal antibody that binds to the domainIV of the extracellular segment of the HER2/neu receptor. The HER2 gene(also known as HER2/neu and ErbB2 gene) is amplified in 20-30% ofearly-stage breast cancers, which makes it overexpressed. Also, incancer, HER2 may send signals without mitogens arriving and binding toany receptor, making it overactive.

HER2 extends through the cell membrane, and carries signals from outsidethe cell to the inside. In healthy people, signaling compounds calledmitogens arrive at the cell membrane, and bind to the outside part ofother members of the HER family of receptors. Those bound receptors thenlink (dimerize) with HER2, activating it. HER2 then sends a signal tothe inside of the cell. The signal passes through different biochemicalpathways. This includes the PI3K/Akt pathway and the MAPK pathway. Thesesignals promote invasion, survival and growth of blood vessels(angiogenesis) of cells.

Cells treated with trastuzumab undergo arrest during the G1 phase of thecell cycle so there is reduced proliferation. It has been suggested thattrastuzumab induces some of its effect by downregulation of HER2/neuleading to disruption of receptor dimerization and signaling through thedownstream PI3K cascade. P27Kip1 is then not phosphorylated and is ableto enter the nucleus and inhibit cdk2 activity, causing cell cyclearrest. Also, trastuzumab suppresses angiogenesis by both induction ofantiangiogenic factors and repression of proangiogenic factors. It isthought that a contribution to the unregulated growth observed in cancercould be due to proteolytic cleavage of HER2/neu that results in therelease of the extracellular domain. Trastuzumab has been shown toinhibit HER2/neu ectodomain cleavage in breast cancer cells.

VIII. Cellular Uptake of Non-Natural Amino Acids

Non-natural amino acid uptake by a eukaryotic cell is one issue that istypically considered when designing and selecting non-natural aminoacids, including but not limited to, for incorporation into a protein.For example, the high charge density of α-amino acids suggests thatthese compounds are unlikely to be cell permeable. Natural amino acidsare taken up into the eukaryotic cell via a collection of protein-basedtransport systems. A rapid screen can be done which assesses whichnon-natural amino acids, if any, are taken up by cells (examples 15 & 16herein illustrate non-limiting examples of tests which can be done onnon-natural amino acids). See, e.g., the toxicity assays in, e.g., theU.S. Patent Publication No. 2004/198637 entitled “Protein Arrays,” whichis herein incorporated by reference in its entirety, and Liu, D. R. &Schultz, P. G. (1999) Progress toward the evolution of an organism withan expanded genetic code. PNAS United States 96:4780-4785. Althoughuptake is easily analyzed with various assays, an alternative todesigning non-natural amino acids that are amenable to cellular uptakepathways is to provide biosynthetic pathways to create amino acids invivo.

Typically, the non-natural amino acid produced via cellular uptake asdescribed herein is produced in a concentration sufficient for efficientprotein biosynthesis, including but not limited to, a natural cellularamount, but not to such a degree as to affect the concentration of theother amino acids or exhaust cellular resources. Typical concentrationsproduced in this manner are about 10 mM to about 0.05 mM.

IX. Biosynthesis of Non-Natural Amino Acids

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular non-natural amino acid may not exist in nature, including butnot limited to, in a cell, the methods and compositions described hereinprovide such methods. For example, biosynthetic pathways for non-naturalamino acids can be generated in host cell by adding new enzymes ormodifying existing host cell pathways. Additional new enzymes includenaturally occurring enzymes or artificially evolved enzymes. Forexample, the biosynthesis of p-aminophenylalanine (as presented in anexample in WO 2002/085923 entitled “In vivo incorporation of unnaturalamino acids”) relies on the addition of a combination of known enzymesfrom other organisms. The genes for these enzymes can be introduced intoa eukaryotic cell by transforming the cell with a plasmid comprising thegenes. The genes, when expressed in the cell, provide an enzymaticpathway to synthesize the desired compound. Examples of the types ofenzymes that are optionally added are provided herein. Additionalenzymes sequences are found, for example, in Genbank. Artificiallyevolved enzymes can be added into a cell in the same manner. In thismanner, the cellular machinery and resources of a cell are manipulatedto produce non-natural amino acids.

A variety of methods are available for producing novel enzymes for usein biosynthetic pathways or for evolution of existing pathways. Forexample, recursive recombination, including but not limited to, asdeveloped by Maxygen, Inc. (available on the world wide web atwww.maxygen.com), can be used to develop novel enzymes and pathways.See, e.g., Stemmer (1994), Rapid evolution of a protein in vitro by DNAshuffling, Nature 370(4):389-391; and, Stemmer, (1994), DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution, Proc. Natl. Acad. Sci. USA., 91:10747-10751.Similarly DesignPath™, developed by Genencor (available on the worldwide web at genencor.com) is optionally used for metabolic pathwayengineering, including but not limited to, to engineer a pathway tocreate a non-natural amino acid in a cell. This technology reconstructsexisting pathways in host organisms using a combination of new genes,including but not limited to those identified through functionalgenomics, molecular evolution and design. Diversa Corporation (availableon the world wide web at diversa.com) also provides technology forrapidly screening libraries of genes and gene pathways, including butnot limited to, to create new pathways for biosynthetically producingnon-natural amino acids.

Typically, the non-natural amino acid produced with an engineeredbiosynthetic pathway as described herein is produced in a concentrationsufficient for efficient protein biosynthesis, including but not limitedto, a natural cellular amount, but not to such a degree as to affect theconcentration of the other amino acids or exhaust cellular resources.Typical concentrations produced in vivo in this manner are about 10 mMto about 0.05 mM. Once a cell is transformed with a plasmid comprisingthe genes used to produce enzymes desired for a specific pathway and anon-natural amino acid is generated, in vivo selections are optionallyused to further optimize the production of the non-natural amino acidfor both ribosomal protein synthesis and cell growth.

X. Additional Synthetic Methodology

The non-natural amino acids described herein may be synthesized usingmethodologies described in the art or using the techniques describedherein or by a combination thereof. As an aid, the following tableprovides various starting electrophiles and nucleophiles which may becombined to create a desired functional group. The information providedis meant to be illustrative and not limiting to the synthetic techniquesdescribed herein.

TABLE 2 Examples of Covalent Linkages and Precursors Thereof CovalentLinkage Product Electrophile Nucleophile Carboxamides Activated estersamines/anilines Carboxamides acyl azides amines/anilines Carboxamidesacyl halides amines/anilines Esters acyl halides alcohols/phenols Estersacyl nitriles alcohols/phenols Carboxamides acyl nitrilesamines/anilines Imines Aldehydes amines/anilines Hydrazones aldehydes orketones Hydrazines Oximes aldehydes or ketones Hydroxylamines Alkylamines alkyl halides amines/anilines Esters alkyl halides carboxylicacids Thioethers alkyl halides Thiols Ethers alkyl halidesalcohols/phenols Thioethers alkyl sulfonates Thiols Esters alkylsulfonates carboxylic acids Ethers alkyl sulfonates alcohols/phenolsEsters Anhydrides alcohols/phenols Carboxamides Anhydridesamines/anilines Thiophenols aryl halides Thiols Aryl amines aryl halidesAmines Thioethers Azindines Thiols Boronate esters Boronates GlycolsCarboxamides carboxylic acids amines/anilines Esters carboxylic acidsAlcohols hydrazines Hydrazides carboxylic acids N-acylureas orAnhydrides carbodiimides carboxylic acids Esters diazoalkanes carboxylicacids Thioethers Epoxides Thiols Thioethers haloacetamides ThiolsAmmotriazines halotriazines amines/anilines Triazinyl ethershalotriazines alcohols/phenols Amidines imido esters amines/anilinesUreas Isocyanates amines/anilines Urethanes Isocyanates alcohols/phenolsThioureas isothiocyanates amines/anilines Thioethers Maleimides ThiolsPhosphite esters phosphoramidites Alcohols Silyl ethers silyl halidesAlcohols Alkyl amines sulfonate esters amines/anilines Thioetherssulfonate esters Thiols Esters sulfonate esters carboxylic acids Etherssulfonate esters Alcohols Sulfonamides sulfonyl halides amines/anilinesSulfonate esters sulfonyl halides phenols/alcohols

In general, carbon electrophiles are susceptible to attack bycomplementary nucleophiles, including carbon nucleophiles, wherein anattacking nucleophile brings an electron pair to the carbon electrophilein order to form a new bond between the nucleophile and the carbonelectrophile.

Non-limiting examples of carbon nucleophiles include, but are notlimited to alkyl, alkenyl, aryl and alkynyl Grignard, organolithium,organozinc, alkyl-, alkenyl, aryl- and alkynyl-tin reagents(organostannanes), alkyl-, alkenyl-, aryl- and alkynyl-borane reagents(organoboranes and organoboronates); these carbon nucleophiles have theadvantage to of being kinetically stable in water or polar organicsolvents. Other non-limiting examples of carbon nucleophiles includephosphorus ylids, enol and enolate reagents; these carbon nucleophileshave the advantage of being relatively easy to generate from precursorswell known to those skilled in the art of synthetic organic chemistry.Carbon nucleophiles, when used in conjunction with carbon electrophiles,engender new carbon-carbon bonds between the carbon nucleophile andcarbon electrophile.

Non-limiting examples of non-carbon nucleophiles suitable for couplingto carbon electrophiles include but are not limited to primary andsecondary amines, thiols, thiolates, and thioethers, alcohols,alkoxides, azides, semicarbazides, and the like. These non-carbonnucleophiles, when used in conjunction with carbon electrophiles,typically generate heteroatom linkages (C—X—C), wherein X is ahetereoatom, including, but not limited to, oxygen, sulfur, or nitrogen.

EXAMPLES Example 1: Synthesis of Compound 1

Compound 1-3: Tetra (ethylene glycol) 1-1 (10 g, 51.5 mmol),N-hydroxyphthalimide 1-2 (8.4 g, 51.15 mmol) and triphenylphosphine(17.6 g, 67 mmol) were dissolved in 300 mL of tetrahydrofuran followedby addition of DIAD (12.8 mL, 61.78 mmol) at 0° C. The resultingsolution was stirred at room temperature overnight, and thenconcentrated to dryness. The residue was purified by flash columnchromatography to give 5.47 g (31%) of compound 1-3.

Compound 1-4: To a solution of compound 1-3 (200 mg, 0.59 mmol) in 15 mLdichloromethane was added Dess-Martin Periodinane (300 mg, 0.71 mmol).The reaction mixture was stirred at ambient temperature overnight. Thereaction was quenched with the solution of sodium bisulfite in 15 mL ofsaturated sodium bicarbonate. The mixture was separated. The organiclayer was washed with saturated sodium bicarbonate, brine, dried oversodium sulfate, filtered and concentrated in vacuo. The residue waspurified by flash column chromatography to give 150 mg (75%) of compound1-4.

Compound 1-6: To a solution of monomethyldolastatin hydrochloride salt1-5 (50 mg, 0.062 mmol) in 1 mL of DMF was added compound 1-4 (63 mg,0.186 mmol) and 70 μL of acetic acid, followed by addition of 8 mg ofsodium cyanoborohydride. The resulting mixture was stirred at ambienttemperature for 2 hours. The reaction mixture was diluted with water andpurified by HPLC to give 60 mg (80%) of compound 1-6. MS (ESI) m/z 547[M+2H], 1092 [M+H].

1d.

Compound 1: Compound 1-6 (60 mg, 0.05 mmol) was dissolved in 1 mL ofDMF. 32 μL of hydrazine was added. The resulting solution was stirred atambient temperature for 1 hour. The reaction was quenched with 1Nhydrochloride solution. The reaction mixture was purified by HPLC togive 33 mg (55%) of compound 1. MS (ESI) m/z 482 [M+2H], 962 [M+H].

Example 2: Synthesis of Compound 2

Compound 2 was synthesized via a similar synthetic route as described inExample 1. MS (ESI) m/z 460 [M+2H], 918 [M+H].

Example 3: Synthesis of Compound 3

Compound 3 was synthesized via similar synthetic route to Example 1. MS(ESI) m/z 438 [M+2H], 974 [M+H].

Example 4: Synthesis of Compound 4

Compound 4-2: To a solution of Val (OtBu)-OH.HCl 4-1 (1 g, 4.77 mmol)and bromoethanol (304.7 μL, 4.3 mmol) in 10 mL of DMF was added 1.68 mlof DIEA. The reaction mixture was stirred at room temperature for 2days. 4.8 mmol of Boc₂OO was added to the reaction mixture, followed by0.84 mL of DIEA. The reaction mixture was stirred at room temperaturefor 2 days. The reaction mixture was concentrated in vacuo and extractedwith ethyl acetate, and washed with water, brine, dried over sodiumsulfate and concentrated in vacuo. The residue was purified by flashcolumn chromatography to give 0.66 g of compound 4-2.

Compound 4-3: To a solution of compound 4-2 (500 mg, 1.58 mmol),N-hydroxyphthalimide (261 mg, 1.6 mmol) and triphenylphosphine (538 mg,2.05 mmol) in 15 mL THF was added DIAD (394 μL, 1.9 mmol) at at 0° C.The resulting solution was stirred at room temperature overnight, andthen concentrated in vacuo. The residue was purified by flash columnchromatography to give 0.68 g of compound 4-3.

Compound 4-4: Compound 4-3 was dissolved in 15 mL 4N HCl/Dioxane. Thereaction mixture was stirred at room temperature for 2 days andconcentrated in vacuo. The residue was dissolved in DMF and treated withBoc₂O (230 μL, 1 mmol) and DIEA (352 μL, 2 mmol). The reaction mixturewas stirred at room temperature for 2 days. The reaction mixture waspurified by HPLC to give 100 mg of compound 4-4.

Compound 4-5: To a solution of compound Boc-Val-Dil-methylDap-OH in DMFis added phe(OtBu)-OH.HCl, HATU and N-methylmorpholine. The reactionmixture is stirred at room temperature for 4 hours. The reaction mixtureis concentrated in vacuo and extracted with ethyl acetate (100 mL×1, 50mL×2). The organic layer is combined and washed with brine, dried oversodium sulfate and concentrated in vacuo. The residue is purified byflash chromatography. The resulting compound is treated with HCl/EtOACto give compound 4-5.

Compound 4-6: To a solution of compound 4-5 in DMF is added compound4-4, HATU and DIEA. The reaction mixture is stirred at room temperaturefor 4 hours. The reaction mixture is concentrated in vacuo and extractedwith ethyl acetate (100 mL×1, 50 mL×2). The organic layer is combinedand washed with brine, dried over sodium sulfate and concentrated invacuo. The residue is purified by flash chromatography to give compound4-6.

Compound 4-7: Compound 4-6 is dissolved in 15 mL 4N HCl/Dioxane. Thereaction mixture is stirred at room temperature for 2 hours andconcentrated in vacuo to give compound 4-7.

Compound 4-8: To a solution of compound 4-7 in 1 mL of DMF is addedformylaldehyde and acetic acid, followed by addition of sodiumcyanoborohydride. The resulting mixture is stirred at ambienttemperature for 2 hours. The reaction mixture is diluted with water andpurified by HPLC to give compound 4-8.

Compound 4: Compound 4-8 is dissolved in 1 mL of DMF. Hydrazine isadded. The resulting solution is stirred at ambient temperature for 1hour. The reaction is quenched with 1N hydrochloride solution. Thereaction mixture is purified by HPLC to give Compound 4.

Example 5: Synthesis of Compound 5

Compound 4-7 is dissolved in 1 mL of DMF. Hydrazine is added. Theresulting solution is stirred at ambient temperature for 1 hour. Thereaction is quenched with 1N hydrochloride solution. The reactionmixture is purified by HPLC to give Compound 5.

Example 6: Synthesis of Compound 6

Compound 6-2: To a solution of compound 6-1 (500 mg, 0.875 mmol) in 3 mLof DMF was added 283 mg of phenylalanine hydrochloride, 433 mg of HATUand 581 μL of N-methylmorpholine. The reaction mixture was stirred atroom temperature for 4 hours. The reaction mixture was concentrated invacuo and extracted with ethyl acetate (100 mL×1, 50 mL×2). The organiclayer was combined and washed with brine, dried over sodium sulfate andconcentrated in vacuo. The residue was purified by flash chromatographyto give 560 mg (76%) of compound 6-2.

Compound 6-3: Compound 6-2 was dissolved in 15 mL 4N HCl/Dioxane. Thereaction mixture was stirred at room temperature for 2 hours andconcentrated in vacuo to give 511 mg of compound 6-3.

Compound 6-4: To a solution of compound 6-3 (368 mg, 0.55 mmol) in 3 mLof DMF was added 255 mg of Boc-N-methyl valine, 314 mg of HATU and 303μL of N-methylmorpholine. The reaction mixture was stirred at roomtemperature for 4 hours. The reaction mixture was concentrated in vacuoand extracted with ethyl acetate (100 mL×1, 50 mL×2). The organic layerwas combined and washed with brine, dried over sodium sulfate andconcentrated in vacuo. The residue was purified by flash chromatographyto give 370 mg (79%) of compound 6-4.

Compound 6-5: To a solution of compound 6-4 (170 mg) in 10 mL MeOH wasadded Seq of 1N LiOH. The reaction mixture was stirred at roomtemperature for 2 hours. The reaction mixture was acidified by 1NHCl andextracted with ethyl acetate washed with brine, dried over sodiumsulfate and concentrated in vacuo to give 150 mg (90%) of compound 6-5.

Compound 6-6: Compound 6-5 was dissolved in 4N HCl/Dioxane. The reactionmixture was stirred at room temperature for 2 hours and concentrated invacuo and purified by HPLC to give 150 mg of compound 6-6.

Compound 6-7: To a solution of compound 6-6 (50 mg, 0.062 mmol) in 1 mLof DMF was added compound 1-4 (63 mg, 0.186 mmol) and 70 μL of aceticacid, followed by addition of 8 mg of sodium cyanoborohydride. Theresulting mixture was stirred at ambient temperature for 2 hours. Thereaction mixture was diluted with water and purified by HPLC to give 60mg (80%) of compound 6-7.

Compound 6: Compound 6-7 (60 mg, 0.05 mmol) was dissolved in 1 mL ofDMF. 32 μL of hydrazine was added. The resulting solution was stirred atambient temperature for 1 hour. The reaction was quenched with 1Nhydrochloride solution. The reaction mixture was purified by HPLC togive 33 mg (55%) of Compound 6.

Example 7: Synthesis of Compound 7

Compound 7 was synthesized via similar synthetic route to Compound 1. MS(ESI) m/z 440 [M+2H], 879 [M+H].

Example 8: Synthesis of Compound 8

Compound 8 was synthesized via similar synthetic route to Compound 1. MS(ESI) m/z 418 [M+2H], 835 [M+H].

Example 9: Synthesis of Compound 9

Compound 9-1: To a solution of compound Boc-Val-Dil-methylDap-OH in DMFis added 4-(2-Aminoethyl)pyridine, HATU and N-methylmorpholine. Thereaction mixture is stirred at room temperature for 4 hours. Thereaction mixture is concentrated in vacuo and extracted with ethylacetate (100 mL×1, 50 mL×2). The organic layer is combined and washedwith brine, dried over sodium sulfate and concentrated in vacuo. Theresidue is purified by flash chromatography. The resulting compound istreated with HCl/EtOAC to give compound 9-1.

Compound 9-2: To a solution of compound 9-1 in DMF is added compound4-4, HATU and DIEA. The reaction mixture is stirred at room temperaturefor 4 hours. The reaction mixture is concentrated in vacuo and extractedwith ethyl acetate (100 mL×1, 50 mL×2). The organic layer is combinedand washed with brine, dried over sodium sulfate and concentrated invacuo. The residue is purified by flash chromatography to give compound9-2.

Compound 9-3: Compound 9-2 is dissolved in 15 mL 4N HCl/Dioxane. Thereaction mixture is stirred at room temperature for 2 hours andconcentrated in vacuo to give compound 9-3.

Compound 9-4: To a solution of compound 9-3 in 1 mL of DMF is addedformylaldehyde and acetic acid, followed by addition of sodiumcyanoborohydride. The resulting mixture is stirred at ambienttemperature for 2 hours. The reaction mixture is diluted with water andpurified by HPLC to give compound 9-4.

Compound 9: Compound 9-4 is dissolved in 1 mL of DMF. Hydrazine isadded. The resulting solution is stirred at ambient temperature for 1hour. The reaction is quenched with 1N hydrochloride solution. Thereaction mixture is purified by HPLC to give compound 9.

Example 10: Synthesis of Compound 10

Compound 10: Compound 9-3 is dissolved in 1 mL of DMF. Hydrazine isadded. The resulting solution is stirred at ambient temperature for 1hour. The reaction is quenched with 1N hydrochloride solution. Thereaction mixture is purified by HPLC to give Example 10.

Example 11: Synthesis of Compound 11

Compound 11-3: To a solution of tetra (ethylene glycol) 11-1 (40.6 mL,235 mmol) in 100 mL of tetrahedrofuran was added 47 mg of sodium. 12 mLof tert-butylacrylate was added after sodium was dissolved. The reactionmixture was stirred at room temperature for 24 hours. The reactionmixture was concentrated in vacuo and quenched with 2 mL of 1 N HCl. Theresidue was suspended in brine and extracted with ethyl acetate (100mL×1, 50 mL×2). The organic layer was combined and washed with brine,dried over sodium sulfate and concentrated in vacuo to give 6.4 g (23%)of compound 11-3.

Compound 11-5: Compound 11-3 (1.0 g, 3.12 mmol), N-hydroxyphthalimide11-4 (611 mg, 3.744 mmol) and triphenylphosphine (1.23 g, 4.68 mmol)were dissolved in 20 mL of tetrahydrofuran followed by addition of DIAD(0.84 mL, 4.06 mmol) at 0° C. The resulting solution was stirred at roomtemperature overnight, and then concentrated to dryness. The residue waspurified by flash column chromatography using SiliaSep Cartridges (80g), eluting with 0-100% ethyl acetate/hexanes, to give 1.0 g (100%) ofcompound 11-5.

Compound 11-6: Compound 11-5 was dissolved in 15 mL 4N HCl/Dioxane. Thereaction mixture was stirred at room temperature for 2 hours andconcentrated in vacuo to give 1.0 g of compound 11-6.

Compound 11-8: To a solution of 30 mg (0.0372 mmol) ofmonomethyldolastatin hydrochloride, 31 mg (0.0744 mmol) of compound 11-6and 38.2 mg (0.082 mmol) of PyBroP in 1 mL of DMF was added 33 μL (0.186mmol) of diisopropylethylamine. The reaction mixture was stirred at roomtemperature for 5 hours. The reaction mixture was purified by HPLC togive 28 mg (65%) of compound 11-8. MS (ESI) m/z 785 [M+2H], 1164[M+H].

Compound 11: Compound 11-8 (28 mg, 0.024 mmol) was dissolved in 1 mL ofDMF. 23 μL (0.72 mmol) of anhydrous hydrazine was added. The resultingsolution was stirred at room temperature for 1 hour. The reaction wasquenched with 1N hydrochloride solution. The reaction mixture waspurified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at254 nm, to give 20 mg (66%) of Compound 11. MS (ESI) m/z 518 [M+2H],1034[M+H].

Example 12: Synthesis of Compound 12

Compound 12-2: To a solution of compound 12-1 (500 mg, 0.875 mmol) in 3mL of DMF was added 283 mg of phenylalanine hydrochloride, 433 mg ofHATU and 581 μL of N-methylmorpholine. The reaction mixture was stirredat room temperature for 4 hours. The reaction mixture was concentratedin vacuo and extracted with ethyl acetate (100 mL×1, 50 mL×2). Theorganic layer was combined and washed with brine, dried over sodiumsulfate and concentrated in vacuo. The residue was purified by flashchromatography to give 560 mg (76%) of compound 12-2.

Compound 12-3: Compound 12-2 was dissolved in 15 mL 4N HCl/Dioxane. Thereaction mixture was stirred at room temperature for 2 hours andconcentrated in vacuo to give 511 mg of compound 12-3.

Compound 12-4: To a solution of compound 12-3 (368 mg, 0.55 mmol) in 3mL of DMF was added 255 mg of Boc-N-methyl valine, 314 mg of HATU and303 μL of N-methylmorpholine. The reaction mixture was stirred at roomtemperature for 4 hours. The reaction mixture was concentrated in vacuoand extracted with ethyl acetate (100 mL×1, 50 mL×2). The organic layerwas combined and washed with brine, dried over sodium sulfate andconcentrated in vacuo. The residue was purified by flash chromatographyto give 370 mg (79%) of compound 12-4.

Compound 12-5: To a solution of compound 12-4 (170 mg) in 10 mL MeOH wasadded 5 eq of 1N LiOH. The reaction mixture was stirred at roomtemperature for 2 hours. The reaction mixture was acidified by 1NHCl andextracted with ethyl acetate washed with brine, dried over sodiumsulfate and concentrated in vacuo to give 150 mg (90%) of compound 12-5.

Compound 12-6: Compound 12-5 was dissolved in 4N HCl/Dioxane. Thereaction mixture was stirred at room temperature for 2 hours andconcentrated in vacuo and purified by HPLC to give 150 mg of compound12-6.

Compound 12-7: To a solution of compound 12-6 in DMF was addedformylaldehyde (3 eq) and 20 eq of acetic acid, followed by addition of2 eq of sodium cyanoborohydride. The resulting mixture was stirred atambient temperature for 2 hours. The reaction mixture was diluted withwater and purified by HPLC to give compound 12-7.

Compound 12-10: tert-Butyl 2-(2-hydroxyethoxy)ethylcarbamate (2.05 g, 10mmol), N-hydroxyphthalimide (1.8 g, 11 mmol) and triphenylphosphine(3.67 g, 14 mmol) were dissolved in 100 mL of tetrahydrofuran followedby addition of DIAD (2.48 mL, 12 mmol) at 0° C. The resulting solutionwas stirred at room temperature overnight, and then concentrated todryness. The residue was treated with 50 mL of 4N HCl/dioxane. Themixture was stirred at room temperature for 2 hours. The solvent wasremoved in vacuo. The residue was treated with ether, filtered, washedwith ether and dried in vacuo to get 2.6 g (91%) of compound 12-10. MS(ESI) m/z 251 [M+H].

Compound 12-11: To a solution of compound 12-10 (20 mg, 0.026 mmol) in 1mL of DMF was added 11.2 mg of compound 12-10, 15 mg of HATU and 23 μLof DIEA. The reaction mixture was stirred at room temperature for 2hours. The reaction mixture was purified by HPLC to give 20 mg (70%) ofcompound 12-4. MS (ESI) m/z 490 [M+2H], 978[M+H].

Compound 12: Compound 12-11 (20 mg, 0.0183 mmol) was dissolved in 1 mLof DMF. 18 μL (0.56 mmol) of anhydrous hydrazine was added. Theresulting solution was stirred at room temperature for 1 hour. Thereaction was quenched with 1N hydrochloride solution. The reactionmixture was purified by preparative HPLC, eluting with 20-70% CH3CN/H2Oin 20 min at 254 nm, to give 14 mg (72%) of Compound 12. MS (ESI) m/z425 [M+2H], 848[M+H].

Example 13: Synthesis of Compound 13

Compound 13-2: Tert-butyl 6-hydroxyhexanoate 13-1 (1.5 g, 1.97 mmol),N-hydroxyphthalimide (1.42 g, 8.76 mmol) and triphenylphosphine (2.82 g,10.76 mmol) were dissolved in 50 mL of tetrahydrofuran followed byaddition of DIAD (2 mL, 9.564 mmol) at 0° C. The resulting solution wasstirred at room temperature overnight, and then concentrated to dryness.The residue was purified by flash column chromatography to give 2.5 g(95%) of compound 13-2.

Compound 13-3: The compound 13-2 was treated with 15 mL 4N HCl indioxane. The reaction mixture was stirred at ambient temperature for 12hours and concentrated to dryness in vacuo to give 900 mg (100%) ofcompound 13-3.

Compound 13-4: To a solution of compound 13-3 (900 mg, 3.0 mmol) in 10mL of THF was added 397 mg of N-hydroxysuccinimide, followed by adding669 mg of DCC. The reaction mixture was stirred at ambient temperatureovernight and filtered. The filtration was concentrated and treated with10 mL of DCM. The DCM solution was stayed at ambient temperature for 1hour and filtered. The filtration was concentrated and purified by flashcolumn chromatography to give 800 mg (71%) of compound 13-4.

Compound 13-6: The mixture of compound 13-4 (435 mg, 1.16 mmol) andVal-Cit-PABOH 13-5¹ (400 mg, 1.054 mmol) in 12 mL of DMF was stirred atambient temperature for 24 hours. The solvent was removed in vacuo. Theresidue was treated with ether, filtered and washed with ether. Thesolid was dried in vacuo to give 660 mg (98%) of compound 13-6.

Compound 13-7: To the solution of compound 13-6 (200 mg, 0.313 mmol) in6 mL of DMF was added bis(p-nitrophenyl)carbonate (286 mg, 0.94 mmol),followed by addition of 110.2 μL of DIEA. The reaction mixture wasstirred at ambient temperature for 5 hours and concentrated. The residuewas treated with ether and filtered. The collected solid was washed withether, 5% citric acid, water, ether and dried in vacuo to give 210 mg(83%) compound 13-7.

Compound 13-9: To a solution of monomethylauristatin hydrochloride salt13-8 (100 mg, 0.1325 mmol) in 2 mL of DMF was added compound 13-7 (159mg, 0.2 mmol) and 10 mg of HOBt, followed by addition of 35.2 μL ofDIEA. The resulting mixture was stirred at ambient temperature for 2days. The reaction mixture was diluted with water and purified by HPLCto give 93 mg (51%) of compound 13-9. MS (ESI) m/z 692 [M+2H], 1382[M+H].

Compound 13: The compound 13-9 (50 mg, 0.036 mmol) was dissolved in 1 mLof DMF. 23 μL of hydrazine was added. The resulting solution was stirredat ambient temperature for 3 hours. The reaction was quenched with 1Nhydrochloride solution. The reaction mixture was purified by HPLC togive 32 mg (65%) of Compound 13. MS (ESI) m/z 638.5 [M+Na+2H], 1253.3[M+H], 1275.8 [M+Na].

Example 14: Synthesis of Compound 14

Compound 14-3: To a solution of tetra (ethylene glycol) 14-1 (40.6 mL,235 mmol) in 100 mL of tetrahedrofuran was added 47 mg of sodium. 12 mLof tert-butylacrylate was added after sodium was dissolved. The reactionmixture was stirred at room temperature for 24 hours. The reactionmixture was concentrated in vacuo and quenched with 2 mL of 1 N HCl. Theresidue was suspended in brine and extracted with ethyl acetate (100mL×1, 50 mL×2). The organic layer was combined and washed with brine,dried over sodium sulfate and concentrated in vacuo to give 6.4 g (23%)of compound 14-3.

Compound 14-5: Compound 14-3 (1.0 g, 3.12 mmol), N-hydroxyphthalimide14-4 (611 mg, 3.744 mmol) and triphenylphosphine (1.23 g, 4.68 mmol)were dissolved in 20 mL of tetrahydrofuran followed by addition of DIAD(0.84 mL, 4.06 mmol) at 0° C. The resulting solution was stirred at roomtemperature overnight, and then concentrated to dryness. The residue waspurified by flash column chromatography using SiliaSep Cartridges s15(80 g), eluting with 0-100% ethyl acetate/hexanes, to give 1.0 g (100%)of compound 14-5.

Compound 14-6: Compound 14-5 was dissolved in 15 mL 4N HCl/Dioxane. Thereaction mixture was stirred at room temperature for 2 hours andconcentrated in vacuo to give 1.0 g of compound 14-6.

Compound 14-7: To a solution of compound 6 (1.93 g, 4.68 mmol) andN-hydroxysuccinimide (646 mg, 5.616 mmol) in 20 mL of tetrahedrofuranwas added 1.062 g (5.148 mmol) of DCC. The reaction mixture was stirredat room temperature overnight and filtered. The filtration wasconcentrated and purified by flash column chromatography using SiliaSepCartridges (80 g), eluting with 0-100% ethyl acetate/hexanes to give2.37 g (100%) of compound 14-7.

Compound 14-8: Compound 14-8 was made according to the literature(Bioconjugat Chem. 2002, 13 (4), 855-869.)

Compound 14-9: To a solution of compound 14-8 (200 mg, 0.527 mmol) in 2mL of DMF was added 295 mg (0.58 mmol) of compound 14-7. The reactionmixture was stirred at room temperature overnight and concentrated invacuo. The residue was treated with ether, filtered, washed with etherand dried in vacuo to give 402 mg (98%) of compound 14-9.

Compound 14-10: To a solution of compound 14-9 (406 mg, 0.527 mmol) andbis(p-nitrophenol) carbonate (481 mg, 1.58 mmol) in 10 mL of DMF wasadded 0.186 mL (1.054 mmol) of diisopropylethylamine. The reactionmixture was stirred at room temperature for 5 hours. The solvent wasremoved in vacuo. The residue was treated with ether, filtered, washedwith ether, 5% citic acid, water, ether and dried in vacuo to give 350mg (72%) of compound 14-10.

Compound 14-11: To a solution of 50 mg (0.062 mmol) ofmonomethyldolastatin hydrochloride, 87.2 mg (0.093 mmol) of compound14-10 and 4.7 mg (0.031 mmol) of HOBt in 1 mL of DMF was added 22 μL(0.124 mmol) of diisopropylethylamine. The reaction mixture was stirredat room temperature for 16 hours. The reaction mixture was purified byHPLC to give 41 mg (42%) of compound 14-11. MS (ESI) m/z 785 [M+2H].

Compound 14: Compound 14-11 (41 mg, 0.026 mmol) was dissolved in 1 mL ofDMF. 17 μL (0.52 mmol) of anhydrous hydrazine was added. The resultingsolution was stirred at room temperature for 1 hour. The reaction wasquenched with 1N hydrochloride solution. The reaction mixture waspurified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at254 nm, to give 22 mg (58%) of compound 14. MS (ESI) m/z 720 [M+2H].

Example 15: Synthesis of Compound 15

Compound 15-2: To a solution of 50 mg (0.062 mmol) ofmonomethyldolastatin hydrochloride, 75 mg (0.093 mmol) of compound 13-7and 4.7 mg (0.031 mmol) of HOBt in 1 mL of DMF was added 22 μL (0.124mmol) of diisopropylethylamine. The reaction mixture was stirred at roomtemperature for 16 hours. The reaction mixture was purified by HPLC togive 41 mg (42%) of compound 15-2. MS (ESI) m/z 718 [M+2H], 1435 [M+H].

Compound 15-2: Compound 15-2 (41 mg, 0.026 mmol) was dissolved in 1 mLof DMF. 17 μL (0.52 mmol) of anhydrous hydrazine was added. Theresulting solution was stirred at room temperature for 1 hour. Thereaction was quenched with 1N hydrochloride solution. The reactionmixture was purified by preparative HPLC, eluting with 20-70% CH3CN/H2Oin 20 min at 254 nm, to give 22 mg (58%) of example 15. MS (ESI) m/z 653[M+2H], 1305 [M+H].

Example 16: Synthesis of Compound 16

Compound 16-3: To a solution of ethylene glycol 16-1 (13.1 mL, 235 mmol)100 mL of tetrahedrofuran was added 47 mg of sodium. 12 mL oftert-butylacrylate was added after sodium was dissolved. The reactionmixture was stirred at room temperature for 24 hours. The reactionmixture was concentrated in vacuo and quenched with 2 mL of 1 N HCl. Theresidue was suspended in brine and extracted with ethyl acetate (100mL×1, 50 mL×2). The organic layer was combined and washed with brine,dried over sodium sulfate and concentrated in vacuo. The residue waspurified by flash column chromatography to give 5.2 g (24%) of compound16-3.

Compound 16-5: Compound 16-3 (2.0 g, 10.5 mmol), N-hydroxyphthalimide(2.05 g, 12.6 mmol) and triphenylphosphine (3.58 g, 13.65 mmol) weredissolved in 50 mL of tetrahydrofuran followed by addition of DIAD (3.26mL, 15.75 mmol) at 0° C. The resulting solution was stirred at roomtemperature overnight, and then concentrated to dryness. The residue waspurified by flash column chromatography to give compound 16-5.

Compound 16-6: Compound 16-5 was dissolved in 15 mL 4N HCl/Dioxane. Thereaction mixture was stirred at room temperature for 2 hours andconcentrated in vacuo to give compound 16-6.

Compound 16-7: To a solution of compound 16-6 (5.16 mmol) andN-hydroxysuccinimide (722 mg, 6.7 mmol) in 20 mL of tetrahedrofuran wasadded 1.28 g (6.2 mmol) of DCC. The reaction mixture was stirred at roomtemperature overnight and filtered. The filtration was concentrated andpurified by flash column chromatography to give 500 mg of compound 16-7.

Compound 16-8: Compound 16-8 was made according to the literature(Bioconjugat Chem. 2002, 13 (4), 855-869.)

Compound 16-9: To a solution of compound 16-8 (5.0 g, 8.3 mmol) andbis(p-nitrophenol) carbonate (7.6 g, 25 mmol) in 100 mL of DMF was added2.92 mL (16.6 mmol) of diisopropylethylamine. The reaction mixture wasstirred at room temperature for 16 hours. The solvent was removed invacuo. The residue was treated with ether, filtered, washed with ether,5% citic acid, water, ether and dried in vacuo to give 5.0 g (81%) ofcompound 16-9.

Compound 16-10: To a solution of 1.0 g (1.24 mmol) ofmonomethyldolastatin hydrochloride, 1.42 g (1.8575 mmol) of compound16-9 and 95 mg (0.62 mmol) of HOBt in 10 mL of DMF was added 437 μL(2.48 mmol) of diisopropylethylamine. The reaction mixture was stirredat room temperature for 16 hours. The reaction mixture was purified byHPLC to give 1.0 g (58%) of compound 16-10. MS (ESI) m/z 700 [M+2H],1398 [M+H].

Compound 16-11: To a solution of compound 16-10 (1.0 g, 0.715 mmol) in15 mL of tetrahedrofuran was added 5 mL (48 mmol) of diethylamine. Thereaction mixture was stirred at room temperature for 1.5 hours andconcentrated in vacuo. The residue was dissolved in 20 mL of DCM,treated with 200 mL of ether and filtered, wshed with ether and dried invacuo to give 860 mg of compound 16-11. MS (ESI) m/z 589 [M+2H], 1176[M+H].

Compound 16: To a solution of 50 mg (0.0425 mmol) of compound 16-11 in 1mL of DMF was added 32 mg (0.085 mmol) of compound 16-7. The reactionmixture was stirred at room temperature for 16 hours. The HPLC and MSshowed reaction done. 27.2 L (0.85 mmol) of anhydrous hydrazine wasadded to the reaction mixture. The reaction was done in 2 hours. Thereaction mixture was acidified with 1N HCl and purified by HPLC to give40 mg (66%) of compound 16. MS (ESI) m/z 654 [M+2H], 1307[M+H].

Example 17

Compound 17-2: To a solution of compound 17-1 (1.0 g, 4.52 mmol) andN-hydroxysuccinimide (572 mg, 4.97 mmol) in 20 mL of tetrahedrofuran wasadded 1.12 g to (5.424 mmol) of DCC. The reaction mixture was stirred atroom temperature overnight and filtered. The filtration was concentratedto give compound 17-2.

Compound 17: To a solution of 50 mg (0.0425 mmol) of compound 16-11 in 1mL of DMF was added 41 mg (0.1275 mmol) of compound 17-2. The reactionmixture was stirred at room temperature for 16 hours. The HPLC and MSshowed reaction done. 20 μL (0.625 mmol) of anhydrous hydrazine wasadded to the reaction mixture. The reaction was done in 2 hours. Thereaction mixture was acidified with 1N HCl and purified by HPLC to give35 mg (60%) of compound 17. MS (ESI) m/z 625 [M+2H], 1249 [M+H].

Example 18: Synthesis of Compound 18

Compound 18: Compound 18-1 was dissolved in 1 mL of DMF. Anhydrous

Compound 18-1: To a solution of compound 6-6, mg (0.062 mmol) ofcompound 14-10 and HOBt in 1 mL of DMF was added diisopropylethylamine.The reaction mixture was stirred at room temperature for 16 hours. Thereaction mixture was purified by HPLC to give compound 18-1.

Compound 18: Compound 18-1 was dissolved in 1 mL of DMF. Anhydroushydrazine was added. The resulting solution was stirred at roomtemperature for 1 hour. The reaction was quenched with 1N hydrochloridesolution. The reaction mixture was purified by preparative HPLC, elutingwith 20-70% CH3CN/H2O in 20 min at 254 nm, to give compound 18.

Example 19: Synthesis of Compound 19

Compound 19-2: tert-Butyl 2-(2-hydroxyethoxy)ethylcarbamate 13 (2.05 g,10 mmol), N-hydroxyphthalimide (1.8 g, 11 mmol) and triphenylphosphine(3.67 g, 14 mmol) were dissolved in 100 mL of tetrahydrofuran followedby addition of DIAD (2.48 mL, 12 mmol) at 0° C. The resulting solutionwas stirred at room temperature overnight, and then concentrated todryness. The residue was treated with 50 mL of 4N HCl/dioxane. Themixture was stirred at room temperature for 2 hours. The solvent wasremoved in vacuo. The residue was treated with ether, filtered, washedwith ether and dried in vacuo to get 2.6 g (91%) of compound 19-2. MS(ESI) m/z 251 [M+H].

Compound 19-3: To the mixture of compound 19-2 (315 mg, 1.1 mmol),Boc-Lys(Boc)-OH (365 mg, 1 mmol), EDC (382 mg, 2 mmol) and HOBt (306 mg,2 mmol) in 10 mL of DCM was added 1.056 mL (6 mmol) ofdiisopropylethylamine. The reaction mixture was stirred at roomtemperature for 3 hours and extracted with ethyl acetate, washed with 5%citric acid, saturate sodium bicarbonate, brine, dried over sodiumsulfate, filtered and concentrated in vacuo. The residue was purifiedflash column chromatography using SiliaSep Cartridges (40 g), elutingwith 0-100% ethyl acetate/hexanes, to give 405 mg (70%) of compound19-3.

Compound 19-4: Compound 19-3 was dissolved in 15 mL 4N HCl/Dioxane. Thereaction mixture was stirred at room temperature for 2 hours andconcentrated in vacuo to give 315 mg (98%) of compound 19-4. MS (ESI)m/z 379 [M+H].

Compound 19-5: To a solution of compound 14-3 (322 mg, 1 mmol) in 20 mLdichloromethane was added Dess-Martin Periodinane (636 mg, 1.5 mmol).The reaction mixture was stirred at room temperature for 3 hours. Thereaction was quenched with a solution of sodium thiosulfate (1.4 g, 8.85mmol) in 15 mL of saturated sodium bicarbonate. The mixture wasseparated. The organic layer was washed with saturated sodiumbicarbonate, brine, dried over sodium sulfate, filtered and concentratedin vacuo. The residue was purified by flash column chromatography usingSiliaSep Cartridges (40 g), eluting with 0-100% ethyl acetate/hexanes togive 170 mg (53%) of compound 19-5.

Compound 19-6: To a solution of monomethyldolastatin hydrochloride 1.0 g(1.24 mmol) in 20 mL of DMF was added 1.19 g (3.72 mmol) of compound 17followed by 1.4 mL (24.8 mmol) of acetic acid and 156 mg (2.48 mmol) ofsodium cyanoborohydride. The resulting mixture was stirred at roomtemperature for 2 hours. The solvent was removed in vacuo. The residuewas adjusted to pH 8 by sodium bicarbonate and extracted with DCM,washed with brine, dried over sodium sulfate, filtered and concentratedin vacuo. The residue was purified by flash column chromatography usingSiliaSep Cartridges (40 g), eluting with 0-5% methanol/DCM to give 680mg (51%) of compound 19-6. MS (ESI) m/z 538 [M+2H], 1075 [M+H].

Compound 19-7: To a solution of compound 19-6 (680 mg, 0.632 mmol) in 5mL of DCM was added 20 mL of 4N HCl/dioxane. The reaction mixture wasstirred at room temperature for 2 hours and concentrated in vacuo. Theresidue was treated with ether, filtered, washed with ether and dried invacuo to give 660 mg (98%) of compound 19-7. MS (ESI) m/z 510 [M+2H],1019 [M+H].

Compound 19-8: To a solution of compound 19-7 (280 mg, 0.257 mmol),compound 19-4 (38 mg, 0.0857 mmol) and N-methylmorpholine (0.283 mL,2.57 mmol) in 5 mL of N-methylmpyrrolidinone was added 98 mg (0.257mmol) of HATU. The reaction mixture was stirred at room temperature for1 hour. The reaction mixture was purified by HPLC to give 160 mg (71%)of compound 19-8. MS (ESI) m/z 596 [M+4H], 794[M+3H], 1191 [M+2H].

Compound 19: Compound 19-8 (160 mg, 0.0613 mmol) was dissolved in 1.5 mLof DMF. 20 μL (0.613 mmol) of anhydrous hydrazine was added. Theresulting solution was stirred at room temperature for 1 hour. Thereaction was quenched with 1N hydrochloride solution. The reactionmixture was purified by preparative HPLC, eluting with 20-70% CH3CN/H2Oin 20 min at 254 nm, to give 120 mg (75%) of compound 19. MS (ESI) m/z451[M+5H], 563[M+4H], 751 [M+3H], 1126 [M+2H].

Example 20: Synthesis of Compound 20

Compound 20-2: To a solution of tetra (ethylene glycol) 20-1 (8.0 g,41.2 mmol) in 100 mL of tetrahedrofuran was added 1.65 g of sodiumhydride at 0° C. The reaction mixture was stirred at room temperaturefor 30 min. 6.21 g of TBS-Cl was added to this solution. The reactionmixture was stirred at room temperature overnight. The reaction mixturewas concentrated in vacuo and quenched with 2 mL of 1 N HCl. The residuewas suspended in brine and extracted with ethyl acetate (100 mL×1, 50mL×2). The organic layer was combined and washed with brine, dried oversodium sulfate and concentrated in vacuo. The residue was purified byflash column chromatography to give 5.7 g of compound 20-2.

Compound 20-3: To a solution of compound 20-2 (500 mg, 1.62 mmol) in 30mL dichloromethane was added Dess-Martin Periodinane (1.03 g, 2.43mmol). The reaction mixture was stirred at room temperature for 3 hours.The reaction was quenched with a solution of sodium thiosulfate (1.4 g,8.85 mmol) in 15 mL of saturated sodium bicarbonate. The mixture wasseparated. The organic layer was washed with saturated sodiumbicarbonate, brine, dried over sodium sulfate, filtered and concentratedin vacuo. The residue was purified by flash column chromatography togive 400 mg of compound 20-3.

Compound 20-4: To a solution of monomethyldolastatin hydrochloride 213mg (0.263 mmol) in 4 mL of DMF was added 245 mg (0.75 mmol) of compound20-3 followed by 0.303 mL (5 mmol) of acetic acid and 34 mg (0.5 mmol)of sodium cyanoborohydride. The resulting mixture was stirred at roomtemperature for 2 hours. The solvent was removed in vacuo. 3 mL of 60%acetonitrile was added, followed by 0.2 mL of HF.Pyridine at 0° C. Theresulting solution was stirred at room temperature for 2 hours. Theorganic solvent was removed in vacuo. The residue was adjusted to pH 8by sodium bicarbonate and extracted with DCM, washed with brine, driedover sodium sulfate, filtered and concentrated in vacuo. The residue waspurified by flash column chromatography to give 160 mg of compound 20-4.MS (ESI) m/z 474 [M+2H], 947[M+H].

Compound 20-5: To a solution of compound 20-4 (50 mg, 0.062 mmol) in 4mL of DCM was added 0.3 mL of phosgene/toluene at 0° C. The reactionmixture was stirred at 0° C. for 3 hours and concentrated in vacuo fornext step without purification.

Compound 20-6: To a solution of compound 19-4 (7.6 mg, 0.017 mmol) andcompound 20-5 (0.062 mmol) was added 25 μL of diisopropylethylamine. Thereaction mixture was stirred at room temperature for 1 hour. Thereaction mixture was purified by HPLC, eluting with 20-70% CH3CN/H2O in20 min at 254 nm, to give 33 mg of compound 20-6. MS (ESI) m/z582[M+4H], 775[M+3H], 1163[M+2H].

Compound 20: Compound 20-6 (33 mg, 0.014 mmol) was dissolved in 1 mL ofDMF. 14 μL (0.43 mmol) of anhydrous hydrazine was added. The resultingsolution was stirred at room temperature for 1 hour. The reaction wasquenched with 1N hydrochloride solution. The reaction mixture waspurified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at254 nm, to give 10 mg of compound 20. MS (ESI) m/z 549 [M+4H],732[M+3H], 1098[M+2H].

Example 21: Synthesis of Compound 21

Compound 21-2: The mixture of N,N′-Dimethylene diamine 21-1 (5 mL, 46.5mmol) and tert-butyl acrylate 13 mL (116 mmol) was heated at 85° C. for1 hour. Another 13 mL (116 mmol) of tert-butyl acrylate was added. Thereaction mixture was continuely heated at 85° C. for 1 hour and stirredat room temperature overnight. The reaction mixture was concentrated invacuo. The residue wasdiluted with hexanes and purified by flash columnchromatography using SiliaSep Cartridges (120 g), eluting with 0-5%methanol/DCM to give 10.1 g (62%) of compound 21-2. MS (ESI) m/z 345[M+H].

Compound 21-3: To a solution of compound 21-2 (5.0 g, 14.5 mmol) in 50mL of DCM was added 40 mL of 4N HCl/dioxane. The reaction mixture wasstirred at room temperature for 2 days and concentrated in vacuo. Theresidue was treated with ether, filtered, washed with ether and dried invacuo to give 4.3 g (97%) of compound 21-3.

Compound 21-4: To a solution of 166 mg (0.544 mmol) of compound 21-3 and0.15 mL of N-methylmorpholine in 10 mL of N-methylpyrrolidinone wasadded 160 mg of compound 16-11, followed by 0.068 mL (0.408 mmol) ofDECP. The reaction mixture was stirred at room temperature for 1 hour.The reaction mixture was purified by preparative HPLC, eluting with35-70% CH3CN/H2O in 20 min at 254 nm, to give 100 mg (50%) of compound21-4. MS (ESI) m/z 464[M+3H], 696 [M+2H], 1391 [M+H].

Compound 21-5: To a solution of compound 19-4 (11 mg, 0.025 mmol),compound 21-4 (115 mg, 0.077 mmol) and N-methylmorpholine (0.028 mL,0.25 mmol) in 1.5 mL of N-methylmpyrrolidinone was added 29.3 mg (0.077mmol) of HATU. The reaction mixture was stirred at room temperature for1 hour. The reaction mixture was purified by HPLC, eluting with 20-70%CH3CN/H2O in 20 min at 254 nm, to give 60 mg (67%) of compound 21-5. MS(ESI) m/z 625 [M+5H], 781 [M+4H], 1041[M+3H].

Compound 21: Compound 21-5 (60 mg, 0.014 mmol) was dissolved in 1 mL ofDMF. 7 μL (0.21 mmol) of anhydrous hydrazine was added. The resultingsolution was stirred at room temperature for 1 hour. The reaction wasquenched with 1N hydrochloride solution. The reaction mixture waspurified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at254 nm, to give 29 mg (58%) of compound 21. MS (ESI) m/z 599 [M+5H], 749[M+4H], 998 [M+3H].

Example 22: Synthesis of Compound 22

Compound 22-1: To a solution of compound 19-4 (7.6 mg, 0.017 mmol),compound 12-7 (40 mg, 0.051 mmol) and DIEA (0.030 mL, 0.17 mmol) in 2 mLof DMF was added 32 mg (0.085 mmol) of HATU. The reaction mixture wasstirred at room temperature for 2 hour. The reaction mixture waspurified by HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, togive 24 mg (68%) of compound 22-1. MS (ESI) m/z 612 [M+3H], 917 [M+2H],1834[M+H].

Compound 22: Compound 22-1 (24 mg, 0.012 mmol) was dissolved in 1 mL ofDMF. 12 μL (0.36 mmol) of anhydrous hydrazine was added. The resultingsolution was stirred at room temperature for 1 hour. The reaction wasquenched with 1N hydrochloride solution. The reaction mixture waspurified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at254 nm, to give 15 mg (58%) of Example 21. MS (ESI) m/z 569 [M+3H],852[M+2H], 1726[M+2H].

Example 23: Synthesis of Compound 23

Compound 23-1: To a solution of compound 14-3 (4.0 g, 12.4 mmol) and 6.6mL (37.2 mmol) of DIEA in 50 mL of DCM was added 3.31 g oftoluenenesulfonyl chloride at 0° C. The reaction mixture was stirred atroom temperature for 2 days. The reaction mixture was extracted withethyl acetate. The organic layer was combined and washed with 5% citricacid, water, brine, dried over sodium sulfate and concentrated in vacuo.The residue was purified by flash column chromatography to give 3.5 g ofcompound 23-1.

Compound 23-2: To a solution of compound 23-1 (3.5 g, 7.34 mmol) in 20mL DMF was added solium azide (1.44 g, 22.02 mmol). The reaction mixturewas stirred at 50° C. for 2 days. The reaction mixture was extractedwith ethyl acetate. The organic layer was washed with water, brine,dried over sodium sulfate, filtered and concentrated in vacuo. Theresidue was purified by flash column chromatography to give 2.1 g ofcompound 23-2.

Compound 23-3: To a solution of compound 23-2 (2.1 g, 6.05 mmol) in 50mL MeOH was added 400 mg (10%) of Pd—C. The resulting mixture wasstirred at room temperature under 1 atm H₂ for 24 hours. The reactionmixture was filtered and concentrated in vacuo to give 2.1 g of compound23-3. MS (ESI) m/z 322[M+H].

Compound 23-4: To a solution of compound 23-3 (33 mg, 0.102 mmol),compound 12-7 (40 mg, 0.051 mmol) and 54 μL of diisopropylethylamine in1 mL of DMF was added 38 mg of HATU. The reaction mixture was stirred atroom temperature for 4 hours. The reaction mixture was purified by HPLCto give 52 mg of compound 23-4. MS (ESI) m/z 525[M+2H], 1049 [M+H].

Compound 23-5: Compound 23-4 (52 mg, 0.045 mmol) was dissolved in 5 mL4N HCl/Dioxane. The reaction mixture was stirred at room temperature for2 hours and concentrated in vacuo to give 52 mg (100%) of compound 23-5.MS (ESI) m/z 497[M+2H], 993 [M+H].

Compound 23-6: To a solution of compound 19-4 (7.6 mg, 0.017 mmol),compound 23-6 (52 mg, 0.051 mmol) and DIEA (0.030 mL, 0.17 mmol) in 2 mLof DMF was added 32 mg (0.085 mmol) of HATU. The reaction mixture wasstirred at room temperature for 2 hour. The reaction mixture waspurified by HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, togive 26 mg (61%) of compound 23-6. MS (ESI) m/z 583[M+4H], 777[M+3H],1165[M+2H].

Compound 23: Compound 23-6 (26 mg, 0.01 mmol) was dissolved in 1 mL ofDMF. 10 μL (0.31 mmol) of anhydrous hydrazine was added. The resultingsolution was stirred at room temperature for 1 hour. The reaction wasquenched with 1N hydrochloride solution. The reaction mixture waspurified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at254 nm, to give 10 mg (40%) of Example 23. MS (ESI) m/z 550[M+4H],733[M+3H], 1100[M+2H].

TABLE 3 Structures of Compounds 1-23 Example Structure  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

Example 24: Transient Transfection

CHO—S culture is seeded at 0.75×10^6/mL approximately 16 hourspre-transfection in FreeStyle Cho medium. Cells are ready to transfectthe next day when the cell count has reached 1.4-1.6×10^6/mL. When cellsreach target count, 400 mM pAF stock is added to a 1.4 mM final cultureconcentration. PEI/DNA complex is prepared as described: DNA (1.42ug/1×10^6 cells) is dissolved in RPMI (5% (v/v) of total culturevolume), DNA/RPMI mixture is incubated at room temperature for 2minutes, PEI stock (1 mg/mL) is added to DNA solution at a 3:1 ratio (mLPEI/ug DNA), and the mixture is incubated at room temperature for 5 min.The culture is gently added to the mixture and swirled. The flasks aretransferred to a 32° C. incubator. At day 6 post-transfection, a westernblot analysis is performed. At day 7 post-transfection, the supernatantis harvested.

Example 25: CD70-ADC In Vivo Efficacy Study

Anti-tumor efficacy of CD70-2H5-HA119-NCA1 conjugated to NCA1 was testedin a renal cell carcinoma xenograft mouse model. 786-O, human renal cellcarcinoma cells, were transferred as tumor fragment for 2 in vivopassages into nude, female, 5 weeks old mice. The mice, ten per testgroup, were treated with different doses of CD70-2H5-HA119-NCA1 in aformulation of 50 mM histidine, 100 mM NaCl, 5% trehalose, pH 6.0 andmeasured against formulation test vehicle without ADC by IV tailinjection once on day one of the study. Mice were weighed and measuredfor tumor three dimensionally using an electronic caliper. Individualtumor volume was calculated as height×width×length. Mice withvascularized tumors (determined by appearance of the tumors) ofappropriate sizes were randomized into treatment groups and were dosedper individual body weight on Day 33 (three (3) weeks followinginjection with cells). Results from this experiment are shown in FIG. 2.

TABLE 4 Test Groups for CD70 In Vivo Efficacy Study Grp Treatment DoseConcentration Dose Volume Route Schedule 1 Vehicle N/A N/A 5 mL/kg IVOnce, day 1 2 CD70-2H5-HA119-NCA1 1 mg/kg 0.2 mg/mL 5 mL/kg IV Once, day1 3 CD70-2H5-HA119-NCA1 3 mg/kg 0.6 mg/mL 5 mL/kg IV Once, day 1 4CD70-2H5-HA119-NCA1 10 mg/kg 2 mg/mL 5 mL/kg IV Once, day 1 *Ten (10)nude mice per group

Example 26: CD70-ADC in Burkitt Lymphoma Model

Anti-tumor efficacy of ARX-αCD70 unconjugated and ARX-αCD70 conjugatedADC was measured in the Daudi (Burkitt lymphoma) tumor model. Nudefemale mice were used as test subjects, ten per group, and to start, asingle IV injection was given on day 1 to all groups (N=20). There werefive groups, 10 test article and 10 vehicle per group, randomized aftera week.

Second IV Treatment Dose route schedule N injection N Vehicle N/A IVonce, day 1 20 none N/A CD70-2H5-HA119-NCA1 1 mg/kg IV once, day 1 20once, approx 10 = vehicle Day 21 10 = test article CD70-2H5-HA119-NCA1 3mg/kg IV once, day 1 20 once, approx 10 = vehicle Day 21 10 = testarticle CD70-2H5-HA119-NCA1 10 mg/kg IV once, day 1 20 once, approx 10 =vehicle Day 21 10 = test article CD70-2H5-HA119- 10 mg/kg IV once, day 120 once, approx 10 = vehicle unconjugated Day 21 10 = test article

A second injection is administered after 21 days (or when tumors startto re-grow), and non-vehicle groups are split into equal groups of 10each and given a second IV injection.

Mice are weighed and measured for tumor three dimensionally using anelectronic caliper. Individual tumor volume is calculated asheight×width×length. Mice with vascularized tumors (determined byappearance of the tumors) of appropriate sizes are randomized intotreatment groups and are dosed per individual body weight on Day 33(three (3) weeks following injection with cells).

Example 27: CD70-2H5-HA119-NCA1 786-O 96h Cytotoxicity Study

Cytotoxicity of ARX-αCD70 antibody drug conjugates was compared todolastatin in 786-O cells. IC50 was measured and the results from thisexperiment are shown in FIG. 3A.

Example 28: CD70-2H5-HA119-NCA1 Caki-1 96h Cytotoxictty Study

Cytotoxicity of ARX-αCD70 antibody drug conjugates was compared todolastatin in Caki-1 cells. IC50 was measured and the results from thisexperiment are shown in FIG. 3B.

Example 29: Anti-Tumor Activity of Three CD70-AS269 in the 786-OXenograft Model of Kidney Cancer

The 786-O renal cell carcinoma tumor model was used in nude female mice.Ten groups of ten mice each were given 786-O tumor fragments and laterrandomized after a week and a half when tumors grew to between 100-300mm³. The groups, one for each dose level and one for vehicle, weretreated as follows:

-   -   Vehicle,    -   CD70-2H5-HA119-NCA1-IgG1v1 at 0.5, 1 and 3 mg/kg    -   CD70-7F2-HA119-NCA1-IgG1v1 at 0.5, 1 and 3 mg/kg    -   CD70-8A1-HA122-NCA1-IgG1v1 at 0.5, 1 and 3 mg/kg

A single dose injection was given intravenously to all 100 mice on day12 following transplantation, and the results from these experimentsinclude data given in FIGS. 4-6.

Example 30: Human Clinical Trial of CD70-2H5-HA119-NCA1 for Oncology

Human Clinical Trial of the Safety and/or Efficacy ofCD70-2H5-HA119-NCA1 for Oncology

Objective: To compare the safety and pharmacokinetics of administeredcomposition comprising ARX-αCD70-linked dolastatin derivative.

Study Design: This study will be a Phase I, single-center, open-label,randomized dose escalation study followed by a Phase II study in renalcancer patients. Patients should not have had exposure to αCD70-linkeddolastatin derivative prior to the study entry. Patients must not havereceived treatment for their cancer within 2 weeks of beginning thetrial. Treatments include the use of chemotherapy, hematopoietic growthfactors, and biologic therapy such as monoclonal antibodies. Patientsmust have recovered from all toxicities (to grade 0 or 1) associatedwith previous treatment. All subjects are evaluated for safety and allblood collections for pharmacokinetic analysis are collected asscheduled. All studies are performed with institutional ethics committeeapproval and patient consent.

Phase I: Patients receive i.v. CD70-2H5-HA119-NCA1 on days 1, 8, and 15of each 28-day cycle. Doses of CD70-2H5-HA119-NCA1 may be held ormodified for toxicity based on assessments as outlined below. Treatmentrepeats every 28 days in the absence of unacceptable toxicity. Cohortsof 3-6 patients receive escalating doses of CD70-2H5-HA119-NCA1 untilthe maximum tolerated dose (MTD) for CD70-2H5-HA119-NCA1 is determined.The MTD is defined as the dose preceding that at which 2 of 3 or 2 of 6patients experience dose-limiting toxicity. Dose limiting toxicities aredetermined according to the definitions and standards set by theNational Cancer Institute (NCI) Common Terminology for Adverse Events(CTCAE) Version 3.0 (Aug. 9, 2006).

Phase II: Patients receive CD70-2H5-HA119-NCA1 as in phase I at the MTDdetermined in phase I. Treatment repeats every 4 weeks for 2-6 coursesin the absence of disease progression or unacceptable toxicity. Aftercompletion of 2 courses of study therapy, patients who achieve acomplete or partial response may receive an additional 4 courses.Patients who maintain stable disease for more than 2 months aftercompletion of 6 courses of study therapy may receive an additional 6courses at the time of disease progression, provided they meet originaleligibility criteria.

Blood Sampling: serial blood is drawn by direct vein puncture before andafter administration of CD70-2H5-HA119-NCA1. Venous blood samples (5 mL)for determination of serum concentrations are obtained at about 10minutes prior to dosing and at approximately the following times afterdosing: days 1, 8, and 15. Each serum sample is divided into twoaliquots. All serum samples are stored at −20° C. Serum samples areshipped on dry ice.

Pharmacokinetics: Patients undergo plasma/serum sample collection forpharmacokinetic evaluation before beginning treatment and at days 1, 8,and 15. Pharmacokinetic parameters are calculated by model independentmethods on a Digital Equipment Corporation VAX 8600 computer systemusing the latest version of the BIOAVL software. The followingpharmacokinetics parameters are determined: peak serum concentration(C_(max)); time to peak serum concentration (t_(max)); area under theconcentration-time curve (AUC) from time zero to the last blood samplingtime (AUC₀₋₇₂) calculated with the use of the linear trapezoidal rule;and terminal elimination half-life (t_(1/2)), computed from theelimination rate constant. The elimination rate constant is estimated bylinear regression of consecutive data points in the terminal linearregion of the log-linear concentration-time plot. The mean, standarddeviation (SD), and coefficient of variation (CV) of the pharmacokineticparameters are calculated for each treatment. The ratio of the parametermeans (preserved formulation/non-preserved formulation) is calculated.

Patient Response to combination therapy: Patient response is assessedvia imaging with X-ray, CT scans, and MRI, and imaging is performedprior to beginning the study and at the end of the first cycle, withadditional imaging performed every four weeks or at the end ofsubsequent cycles. Imaging modalities are chosen based upon the cancertype and feasibility/availability, and the same imaging modality isutilized for similar cancer types as well as throughout each patient'sstudy course. Response rates are determined using the RECIST criteria.(Therasse et al, J. Natl. Cancer Inst. 2000 Feb. 2; 92(3):205-16;http://ctep.cancer.gov/forms/TherasseRECISTJNCI.pdf). Patients alsoundergo cancer/tumor biopsy to assess changes in progenitor cancer cellphenotype and clonogenic growth by flow cytometry, Western blotting, andIHC, and for changes in cytogenetics by FISH. After completion of studytreatment, patients are followed periodically for 4 weeks.

Additional Assays for Cytotoxic, Cytostatic, and InunnomodulatoryActivities

Methods of determining whether an antibody mediates effector functionagainst a target cell are known. Illustrative, non-limiting examples ofsuch methods are described herein.

For determining whether an anti-CD70 antibody or derivative mediatesantibody-dependent cellular cytotoxicity against activated immune cellsor CD70-expressing cancer cells, an assay that measures target celldeath in the presence of antibody and effector immune cells may be used.An assay used to measure this type of cytotoxicity can be based ondetermination of ⁵¹Cr release from metabolically-labeled targets cellsafter incubation in the presence of effector cells and target-specificantibody (see, e.g., Perussia and Loza, 2000, Methods in MolecularBiology 121:179-92; and “⁵¹Cr Release Assay of Antibody-DependentCell-Mediated Cytotoxicity (ADCC)” in Current Potocols in Immunology,Coligan et al. eds., Wileyand Sons, 1993). For example, activated immunecells (e.g., activated lymphocytes) or CD70-expressing cancer cellslabeled with Na₂ ⁵¹CrO₄ and plated at a density of 5,000 cells per wellof a 96-well plate can be treated with varying concentrations ofanti-CD70 antibody for 30 minutes then mixed with normal humanperipheral blood mononuclear cells (PBMC) for 4 hours. The membranedisruption that accompanies target cell death releases ⁵¹Cr into theculture supernatant which may be collected and assessed forradioactivity as a measure of cytotoxic activity. Other assays tomeasure ADCC may involve nonradioactive labels or be based on inducedrelease of specific enzymes. For example, a non-radioactive assay basedon time-resolved fluorometry is commercially available (Delphia, PerkinElmer). This assay is based on loading target cells with anacetoxymethyl ester of fluorescence enhancing ligand (BATDA) thatpenetrates the cell membrane then hydrolyses to form a membraneimpermeable hydrophilic ligand (TDA). When mixed with target specificantibody and PBMC effector cells, TDA is released from lysed cells andis available to form a highly fluorescent chelate when mixed withEuropium. The signal, measured with a time-resolved fluorometer,correlates with the amount of cell lysis.

To determine whether an anti-CD70 antibody or derivative mediatesantibody-dependent cellular phagocytosis against activated immune cellsor CD70-expressing cancer cells, an assay that measures target cellinternalization by effector immune cells (e.g., fresh culturedmacrophages or established macrophage-like cell line) may be used (see,e.g., Munn and Cheung, 1990, J. Exp. Med. 172:231-37; Keler et al.,2000, J. Immunol. 164:5746-52; Akewanlop et al., 2001, Cancer Res.61:4061-65). For example, target cells may be labeled with a lipophilicmembrane dye such as PKH67 (Sigma), coated with target-specificantibody, and mixed with effector immune cells for 4-24 hours. Theeffector cells may then be identified by counterstaining with afluorochrome-labeled antibody specific for a phagocytic cell surfacemarker (e.g., CD14) and the cells analyzed by two-color flow cytometryor fluoresence microscopy. Dual-positive cells represent effector cellsthat have internalized target cells. For these assays, effector cellsmay be monocytes derived from PBMC that have been differentiated intomacrophages by culture for 5-10 days with M-CSF or GM-CSF (see, e.g.,Munn and Cheung, supra). Human macrophage-like cell lines U937 (Larricket al., 1980, J. Immunology 125:6-12) or THP-1 (Tsuchiya et al., 1980,Int. J. Cancer 26:171-76) which are available from ATCC may be used asan alternative phagocytic cell source.

Methods of determining whether an antibody mediates complement-dependentcytotoxicity upon binding to target cells are also known. The samemethods can be applied to determine whether a CD70-binding agentmediates CDC on activated immune cells or CD70-expressing cancer cells.Illustrative examples of such methods are described infra.

The source of active complement can either be normal human serum orpurified from laboratory animal including rabbits. In a standard assay,a CD70-binding agent is incubated with CD70-expressing activated immunecells (e.g., activated lymphocytes) or CD70-expressing cancer cells inthe presence of complement. The ability of such CD70-binding agent tomediate cell lysis can be determined by several readouts. In oneexample, a Nasup.51CrO₄ release assay is used. In this assay, targetcells are labeled with Na⁵¹CrO₄. Unincorporated Na⁵¹CrO₄ is washed offand cells are plated at a suitable density, typically between 5,000 to50,000 cells/well, in a 96-well plate. Incubation with the CD70-bindingagent in the presence of normal serum or purified complement typicallylast for 2-6 hours at 37.degree. C. in a 5% CO₂ atmosphere. Releasedradioactivity, indicating cell lysis, is determined in an aliquot of theculture supernatant by gamma ray counting. Maximum cell lysis isdetermined by releasing incorporated Na⁵¹CrO₄ by detergent (0.5-1% NP-40or Triton X-100) treatment. Spontaneous background cell lysis isdetermined in wells where only complement is present without anyCD70-binding agents. Percentage cell lysis is calculated as(CD70-binding agent-induced lysis-spontaneous lysis)/maximum cell lysis.The second readout is a reduction of metabolic dyes, e.g., Alamar Blue,by viable cells. In this assay, target cells are incubated withCD70-binding agent with complement and incubated as described above. Atthe end of incubation, 1/10 volume of Alamar Blue (BiosourceInternational, Camarillo, Calif.) is added. Incubation is continued forup to 16 hours at 37.degree. C. in a 5% CO₂ atmosphere. Reduction ofAlamar Blue as an indication of metabolically active viable cells isdetermined by fluorometric analysis with excitation at 530 nm andemission at 590 nm. The third readout is cellular membrane permeabilityto propidium iodide (PI). Formation of pores in the plasma membrane as aresult of complement activation facilitates entry of PI into cells whereit will diffuse into the nuclei and bind DNA. Upon binding to DNA, PIfluorescence in the 600 nm significantly increases. Treatment of targetcells with CD70-binding agent and complement is carried out as describedabove. At end of incubation, PI is added to a final concentration of5.mu.g/ml. The cell suspension is then examined by flow cytometry usinga 488 nm argon laser for excitation. Lysed cells are detected byfluorescence emission at 600 nm.

Additional Animal Models of Immunological Disorders or CD70-ExpressingCancers

The anti-CD70 binding agents of the present invention, e.g., antibodiesor derivatives with one or more non-naturally encoded amino acids andADCs of the invention, can be tested or validated in animal models ofimmunological disorders or CD70-expressing cancers. A number ofestablished animal models of immunological disorders or CD70-expressingcancers are known to the skilled artisan, any of which can be used toassay the efficacy of the anti-CD70 antibody or derivative. Non-limitingexamples of such models are described infra.

Examples for animal models of systemic and organ-specific autoimmunediseases including diabetes, lupus, systemic sclerosis, Sjogren'sSyndrome, experimental autoimmune encephalomyelitis (multiplesclerosis), thyroiditis, myasthenia gravis, arthritis, uveitis, andinflammatory bowel disease have been described by Bigazzi, “AnimalModels of Autoimmunity: Spontaneous and Induced,” in The AutoimmuneDiseases (Rose and Mackay eds., Academic Press, 1998) and in “AnimalModels for Autoimmune and Inflammatory Disease,” in Current Protocols inImmunology (Coligan et al. eds., Wiley and Sons, 1997).

Allergic conditions, e.g., asthma and dermatitis, can also be modeled inrodents. Airway hypersensitivity can be induced in mice by ovalbumin(Tomkinson et al., 2001, J. Immunol. 166:5792-800) or Schistosomamansoni egg antigen (Tesciuba et al., 2001, J. Immunol. 167:1996-2003).The Nc/Nga strain of mice show marked increase in serum IgE andspontaneously develop atopic dermatitis-like leisons (Vestergaard etal., 2000, Mol. Med. Today 6:209-10; Watanabe et al., 1997, Int.Immunol. 9:461-66; Saskawa et al., 2001, Int. Arch. Allergy Immunol.126:239-47).

Injection of immuno-competent donor lymphocytes into a lethallyirradiated histo-incompatible host is a classical approach to induceGVHD in mice. Alternatively, the parent B6D2F1 murine model provides asystem to induce both acute and chronic GVHD. In this model the B6D2F1mice are F1 progeny from a cross between the parental strains of C57BL/6and DBA/2 mice. Transfer of DBA/2 lymphoid cells into non-irradiatedB6D2F1 mice causes chronic GVHD, whereas transfer of C57BL/6, C57BL/10or B10.D2 lymphoid cells causes acute GVHD (Slayback et al., 2000, BoneMarrow Transpl. 26:931-938; Kataoka et al., 2001, Immunology103:310-318).

Additionally, both human hematopoietic stem cells and mature peripheralblood lymphoid cells can be engrafted into SCID mice, and these humanlympho-hematopoietic cells remain functional in the SCID mice (McCune etal., 1988, Science 241:1632-1639; Kamel-Reid and Dick, 1988, Science242:1706-1709; Mosier et al., 1988, Nature 335:256-259). This hasprovided a small animal model system for the direct testing of potentialtherapeutic agents on human lymphoid cells. (See, e.g., Tournoy et al.,2001, J. Immunol. 166:6982-6991).

Moreover, small animal models to examine the in vivo efficacies of theanti-CD70 antibodies or derivatives can be created by implantingCD70-expressing human tumor cell lines into appropriate immunodeficientrodent strains, e.g., athymic nude mice or SCID mice. Examples ofCD70-expressing human lymphoma cell lines include, for example, Daudi(Ghetie et al., 1994, Blood 83:1329-36; Ghetie et al., 1990, Int. J.Cancer 15:481-85; de Mont et al., 2001, Cancer Res. 61:7654-59),HS-Sultan (Cattan and Maung, 1996, Cancer Chemother. Pharmacol.38:548-52; Cattan and Douglas, 1994, Leuk. Res. 18:513-22), Raji(Ochakovskaya et al., 2001, Clin. Cancer Res. 7:1505-10; Breisto et al.,1999, Cancer Res. 59:2944-49), and CA46 (Kreitman et al., 1999, Int. J.Cancer 81:148-55). Non-limiting example of a CD70-expressing Hodgkin'slymphoma line is L428 (Drexler, 1993, Leuk. Lymphoma 9:1-25; Dewan etal., 2005, Cancer Sci. 96:466-473). Non-limiting examples of CD70expressing human renal cell carcinoma cell lines include 786-O (Ananthet al., 1999, Cancer Res. 59:2210-16; Datta et al., 2001, Cancer Res.61:1768-75), ACHN (Hara et al., 2001, J. Urol. 166:2491-94; Miyake etal., 2002, J. Urol. 167:2203-08), Caki-1 (Prewett et al., 1998, Clin.Cancer Res. 4:2957-66; Shi and Siemann, 2002, Br. J. Cancer 87:119-26),and Caki-2 (Zellweger et al., 2001, Neoplasia 3:360-67). Non-limitingexamples of CD70-expressing nasopharyngeal carcinoma cell lines includeC15 and C17 (Busson et al., 1988, Int. J. Cancer 42:599-606; Bernheim etal., 1993, Cancer Genet. Cytogenet. 66:11-5). Non-limiting examples ofCD70-expressing human glioma cell lines include U373 (Palma et al.,2000, Br. J. Cancer 82:480-7) and U87MG (Johns et al., 2002, Int. J.Cancer 98:398-408). Non-limiting examples of multiple myeloma cell linesinclude MM.1S (Greenstein et al., 2003, Experimental Hematology31:271-282) and L363 (Diehl et al., 1978, Blut 36:331-338). (See alsoDrexler and Matsuo, 2000, Leukemia Research 24:681-703). These tumorcell lines can be established in immunodeficient rodent hosts either assolid tumor by subcutaneous injections or as disseminated tumors byintravenous injections. Once established within a host, these tumormodels can be applied to evaluate the therapeutic efficacies of theanti-CD70 antibody or derivatives as described herein on modulating invivo tumor growth.

CD70-Associated Disorders

The anti-CD70 antibodies and ADCs described herein are useful fortreating or preventing a CD70-expressing cancer or an immunologicaldisorder characterized by expression of CD70 by inappropriate activationof immune cells (e.g., lymphocytes or dendritic cells). Such expressionof CD70 can be due to, for example, increased CD70 protein levels on thecells surface and/or altered antigenicity of the expressed CD70.Treatment or prevention of the immunological disorder, according to themethods described herein, is achieved by administering to a subject inneed of such treatment or prevention an effective amount of theanti-CD70 antibody or derivative, whereby the antibody or derivative (i)binds to activated immune cells that express CD70 and that areassociated with the disease state and (ii) exerts a cytotoxic,cytostatic, or immunomodulatory effect on the activated immune cells. Insome embodiments, the cytotoxic, cytostatic, or immunomodulatory isexerted without conjugation to a cytotoxic, cytostatic, orimmunomodulatory agent. In some embodiments, the cytotoxic, cytostatic,or immunomodulatory is exerted by conjugation to a cytotoxic,cytostatic, or immunomodulatory agent.

Immunological diseases that are characterized by inappropriateactivation of immune cells and that can be treated or prevented by themethods described herein can be classified, for example, by the type(s)of hypersensitivity reaction(s) that underlie the disorder. Thesereactions are typically classified into four types: anaphylacticreactions, cytotoxic (cytolytic) reactions, immune complex reactions, orcell-mediated immunity (CMI) reactions (also referred to as delayed-typehypersensitivity (DTH) reactions). (See, e.g., Fundamental Immunology(William E. Paul ed., Raven Press, N.Y., 3rd ed. 1993).)

Specific examples of such immunological diseases include the following:rheumatoid arthritis, psoriatic arthritis, autoimmune demyelinativediseases (e.g., multiple sclerosis, allergic encephalomyelitis),endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus,myasthenia gravis, Grave's disease, glomerulonephritis, autoimmunehepatological disorder, inflammatory bowel disease (e.g., Crohn'sdisease), anaphylaxis, allergic reaction, Sjogren's syndrome, type Idiabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis,fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure,Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis,thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease,pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis,atherosclerosis, subacute cutaneous lupus erythematosus,hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia,idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigusvulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata,pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome(calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyl), and telangiectasia), male and female autoimmuneinfertility, ankylosing spondolytis, ulcerative colitis, mixedconnective tissue disease, polyarteritis nedosa, systemic necrotizingvasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome,Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrentabortion, anti-phospholipid syndrome, farmer's lung, erythemamultiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmunechronic active hepatitis, bird-fancier's lung, toxic epidermalnecrolysis, Alport's syndrome, alveolitis, allergic alveolitis,fibrosing alveolitis, interstitial lung disease, erythema nodosum,pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis,polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cellarteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema,lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome,Kawasaki's disease, dengue, encephalomyelitis, endocarditis,endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum,psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman'ssyndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis,heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy,Henoch-Schonlein purpura, graft versus host disease, transplantationrejection, cardiomyopathy, Eaton-Lambert syndrome, relapsingpolychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan'ssyndrome, and autoimmune gonadal failure.

Accordingly, the methods described herein encompass treatment ofdisorders of B lymphocytes (e.g., systemic lupus erythematosus,Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes),Th₁-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis,psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave'sdisease, primary biliary cirrhosis, Wegener's granulomatosis,tuberculosis, or graft versus host disease), or Th₂-lymphocytes (e.g.,atopic dermatitis, systemic lupus erythematosus, atopic asthma,rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemicsclerosis, or chronic graft versus host disease). Generally, disordersinvolving dendritic cells involve disorders of Th₁-lymphocytes orTh₂-lymphocytes.

In some embodiments, the immunological disorder is a T cell-mediatedimmunological disorder, such as a T cell disorder in which activated Tcells associated with the disorder express CD70. Anti-CD70 bindingagents (e.g., antibodies or derivatives) can be administered to depletesuch CD70-expressing activated T cells. In a specific embodiment,administration of anti-CD70 antibodies or derivatives can depleteCD70-expressing activated T cells, while resting T cells are notsubstantially depleted by the anti-CD70 or derivative. In this context,“not substantially depleted” means that less than about 60%, or lessthan about 70% or less than about 80% of resting T cells are notdepleted.

The anti-CD70 antibodies and ADCs described herein are also useful fortreating or preventing a CD70-expressing cancer. Treatment or preventionof a CD70-expressing cancer, according to the methods described herein,is achieved by administering to a subject in need of such treatment orprevention an effective amount of the anti-CD70 antibody or derivative,whereby the antibody or derivative (i) binds to CD70-expressing cancercells and (ii) exerts a cytotoxic or cytostatic effect to deplete orinhibit the proliferation of the CD70-expressing cancer cells. In someembodiments, the cytotoxic, cytostatic, or immunomodulatory is exertedwithout conjugation to a cytotoxic, cytostatic, or immunomodulatoryagent. In some embodiments, the cytotoxic, cytostatic, orimmunomodulatory is exerted by conjugation to a cytotoxic, cytostatic,or immunomodulatory agent.

CD70-expressing cancers that can be treated or prevented by the methodsdescribed herein include, for example, different subtypes ofNon-Hodgkin's Lymphoma (indolent NHLs, follicular NHLs, smalllymphocytic lymphomas, lymphoplasmacytic NHLs, or marginal zone NHLs);Hodgkin's disease (e.g., Reed-Sternberg cells); cancers of the B-celllineage, including, e.g., diffuse large B-cell lymphomas, follicularlymphomas, Burkitt's lymphoma, mantle cell lymphomas, B-cell lymphocyticleukemias (e.g., acute lymphocytic leukemia, chronic lymphocyticleukemia); Epstein Barr Virus positive B cell lymphomas; renal cellcarcinomas (e.g., clear cell and papillary); nasopharyngeal carcinomas;thymic carcinomas; gliomas; glioblastomas; neuroblastomas; astrocytomas;meningiomas; Waldenstrom macroglobulinemia; multiple myelomas; andcolon, stomach, and rectal carcinomas. The cancer can be, for example,newly diagnosed, pre-treated or refractory or relapsed. In someembodiments, a CD70-expressing cancer has at least about 15,000, atleast about 10,000 or at least about 5,000 CD70 molecules/cell.

With respect to therapeutic regimens for combinatorial administration ofconjugated or unconjugated anti-CD70 antibodies of the presentinvention, in a specific embodiment, an anti-CD70 binding agent isadministered concurrently with a therapeutic agent. In another specificembodiment, the anti-CD70 antibody is conjugated to a therapeutic agent.In some embodiments, the subject is monitored following administrationof the anti-CD70 binding agent, and optionally the therapeutic agent.

The therapeutic agent can be, for example, any agent that exerts atherapeutic effect on cancer cells or activated immune cells. Typically,the therapeutic agent is a cytotoxic or immunomodulatory agent. Suchcombinatorial administration can have an additive or synergistic effecton disease parameters (e.g., severity of a symptom, the number ofsymptoms, or frequency of relapse).

Useful classes of cytotoxic or immunomodulatory agents include, forexample, antitubulin agents, auristatins, DNA minor groove binders, DNAreplication inhibitors, alkylating agents (e.g., platinum complexes suchas cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinumcomplexes and carboplatin), anthracyclines, antibiotics, antifolates,antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides,fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas,platinols, pre-forming compounds, purine antimetabolites, puromycins,radiation sensitizers, steroids, taxanes, topoisomerase inhibitors,vinca alkaloids, and the like.

Individual cytotoxic or immunomodulatory agents include, for example, anandrogen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine,bleomycin, busulfan, buthionine sulfoximine, camptothecin, carboplatin,carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine,cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B,dacarbazine, dactinomycin (actinomycin), daunorubicin, decarbazine,docetaxel, doxorubicin, an estrogen, 5-fluordeoxyuridine,5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide,irinotecan, lomustine (CCNU), mechlorethamine, melphalan,6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone,nitroimidazole, paclitaxel, plicamycin, procarbizine, rapamycin(Sirolimus), streptozotocin, tenoposide, 6-thioguanine, thioTEPA,topotecan, vinblastine, vincristine, vinorelbine, VP-16 and VM-26.

In some typical embodiments, the therapeutic agent is a cytotoxic agent.Suitable cytotoxic agents include, for example, dolastatins (e.g.,auristatin E, AFP, MMAF, MMAE), DNA minor groove binders (e.g.,enediynes and lexitropsins), duocarmycins, taxanes (e.g., paclitaxel anddocetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan,morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin,echinomycin, combretastatin, netropsin, epothilone A and B,estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide,eleutherobin, and mitoxantrone.

In some embodiments, the cytotoxic agent is a conventionalchemotherapeutic such as, for example, doxorubicin, paclitaxel,melphalan, vinca alkaloids, methotrexate, mitomycin C or etoposide. Insome embodiments, the therapeutic agent can be a combined therapy, suchas CHOP (Cyclophosphamide, Doxorubicin, Prednisolone and Vincristine),CHOP-R (Cyclophosphamide, Doxorubicin Vincristine, Prednisolone, andrituximab) or ABVD (Doxorubicin, Bleomycin, Vinblastine andDacarbazine). Agents such as CC-1065 analogues, calicheamicin,maytansine, analogues of dolastatin 10, rhizoxin, and palytoxin can belinked to the anti-CD70 antibodies or derivatives thereof.

In specific embodiments, the cytotoxic or cytostatic agent is auristatinE (also known in the art as dolastatin-10) or a derivative thereof.Typically, the auristatin E derivative is, e.g., an ester formed betweenauristatin E and a keto acid. For example, auristatin E can be reactedwith paraacetyl benzoic acid or benzoylvaleric acid to produce AEB andAEVB, respectively. Other typical auristatin derivatives include AFP,MMAF, and MMAE. The synthesis and structure of auristatin E and itsderivatives are described in U.S. Patent Application Publication Nos.20030083263 and 20050009751), International Patent Application No.PCT/US03/24209, International Patent Application No. PCT/US02/13435, andU.S. Pat. Nos. 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860;5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284;5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744;4,879,278; 4,816,444; and 4,486,414.

In specific embodiments, the cytotoxic agent is a DNA minor groovebinding agent. (See, e.g., U.S. Pat. No. 6,130,237.) For example, insome embodiments, the minor groove binding agent is a CBI compound. Inother embodiments, the minor groove binding agent is an enediyne (e.g.,calicheamicin). Examples of anti-tubulin agents include, but are notlimited to, taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)),T67 (Tularik), vinca alkyloids (e.g., vincristine, vinblastine,vindesine, and vinorelbine), and dolastatins (e.g., auristatin E, AFP,MMAF, MMAE, AEB, AEVB). Other antitubulin agents include, for example,baccatin derivatives, taxane analogs (e.g., epothilone A and B),nocodazole, colchicine and colcimid, estramustine, cryptophysins,cemadotin, maytansinoids, combretastatins, discodermolide, andeleutherobin.

In some embodiments, the cytotoxic agent is a maytansinoid, anothergroup of anti-tubulin agents. For example, in specific embodiments, themaytansinoid is maytansine or DM-1 (ImmunoGen, Inc.; see also Chari etal., 1992, Cancer Res. 52:127-131). In some embodiments, the therapeuticagent is not a radioisotope. In some embodiments, the therapeutic agentis not ricin or saporin. In certain embodiments, the therapeutic agentis an anti-VEGF agent, such as AVASTIN (bevacizumab) or NEXAVAR(Sorafenib); a PDOF blocker, such as SUTENT (sunitinib malate); or akinase inhibitor, such as NEXAVAR (sorafenib tosylateor). In someembodiments, the cytotoxic or immunomodulatory agent is anantimetabolite. The antimetabolite can be, for example, a purineantagonist (e.g., azothioprine or mycophenolate mofetil), adihydrofolate reductase inhibitor (e.g., methotrexate), acyclovir,gangcyclovir, zidovudine, vidarabine, ribavarin, azidothymidine,cytidine arabinoside, amantadine, dideoxyuridine, iododeoxyuridine,poscarnet, or trifluridine.

In other embodiments, the cytotoxic or immunomodulatory agent istacrolimus, cyclosporine or rapamycin. In further embodiments, thecytoxic agent is aldesleukin, alemtuzumab, alitretinoin, allopurinol,altretamine, amifostine, anastrozole, arsenic trioxide, bexarotene,bexarotene, calusterone, capecitabine, celecoxib, cladribine,Darbepoetin alfa, Denileukin diftitox, dexrazoxane, dromostanolonepropionate, epirubicin, Epoetin alfa, estramustine, exemestane,Filgrastim, floxuridine, fludarabine, fulvestrant, gemcitabine,gemtuzumab ozogamicin, goserelin, idarubicin, ifosfamide, imatinibmesylate, Interferon alfa-2a, irinotecan, letrozole, leucovorin,levamisole, meclorethamine or nitrogen mustard, megestrol, mesna,methotrexate, methoxsalen, mitomycin C, mitotane, nandrolonephenpropionate, oprelvekin, oxaliplatin, pamidronate, pegademase,pegaspargase, pegfilgrastim, pentostatin, pipobroman, plicamycin,porfimer sodium, procarbazine, quinacrine, rasburicase, Sargramostim,streptozocin, tamoxifen, temozolomide, teniposide, testolactone,thioguanine, toremifene, Tositumomab, Trastuzumab, tretinoin, uracilmustard, valrubicin, vinblastine, vincristine, vinorelbine orzoledronate.

In additional embodiments, the therapeutic agent is an antibody, such asa humanized anti HER2 monoclonal antibody, RITUXAN (rituximab;Genentech; a chimeric anti CD20 monoclonal antibody); OVAREX (AltaRexCorporation, MA); PANOREX (Glaxo Wellcome, NC; a murine IgG2a antibody);Cetuximab Erbitux (Imclone Systems Inc., NY; an anti-EGFR IgG chimericantibody); Vitaxin (Medlmmune, Inc., MD; Campath I/H (Leukosite, MA; ahumanized IgG1 antibody); Smart MI95 (Protein Design Labs, Inc., CA; ahumanized anti-CD33 IgG antibody); LymphoCide (Immunomedics, Inc., NJ; ahumanized anti-CD22 IgG antibody); Smart ID10 (Protein Design Labs,Inc., CA; a humanized anti-HLA-DR antibody); Oncolym (Techniclone, Inc.,CA; a radiolabeled murine anti-HLA-Dr10 antibody); Allomune(BioTransplant, CA; a humanized anti-CD2 mAb); Avastin (Genentech, Inc.,CA; an anti-VEGF humanized antibody); Epratuzamab (Immunomedics, Inc.,NJ and Amgen, CA; an anti-CD22 antibody); CEAcide (Immunomedics, NJ; ahumanized anti-CEA antibody); or an anti-CD40 antibody (e.g., asdisclosed in U.S. Pat. No. 6,838,261).

Other suitable antibodies include, but are not limited to, antibodiesagainst the following antigens: CA125, CA15-3, CA19-9, L6, Lewis Y,Lewis .lamda. alpha etoprotein, CA 242, placental alkaline phosphatase,prostate specific membrane antigen, prostatic acid phosphatase,epidermal growth factor, MAGE-1, MAGE-2, MAGE-3, MAGE-4,anti-transferrin receptor, p97, MUC1-KLH, CEA, gp100, MART1, ProstateSpecific Antigen, IL-2 receptor, CD20, CD52, CD30, CD33, CD22, humanchorionic gonadotropin, CD38, CD40, mucin, P21, MPG, and Neu oncogeneproduct.

In some embodiments, the therapeutic agent is an immunomodulatory agent.The immunomodulatory agent can be, for example, gancyclovir, etanercept,tacrolimus, cyclosporine, rapamycin, REVLIMID (lenalidomide),cyclophosphamide, azathioprine, mycophenolate mofetil or methotrexate.Alternatively, the immunomodulatory agent can be, for example, aglucocorticoid (e.g., cortisol or aldosterone) or a glucocorticoidanalogue (e.g., prednisone or dexamethasone).

In some typical embodiments, the immunomodulatory agent is ananti-inflammatory agent, such as arylcarboxylic derivatives,pyrazole-containing derivatives, oxicam derivatives and nicotinic acidderivatives. Classes of anti-inflammatory agents include, for example,cyclooxygenase inhibitors, 5-lipoxygenase inhibitors, and leukotrienereceptor antagonists. In some embodiments, the immodulatory agent is acytokine, such as G-CSF, GM-CSF or IL-2. Suitable cyclooxygenaseinhibitors include meclofenamic acid, mefenamic acid, carprofen,diclofenac, diflunisal, fenbufen, fenoprofen, ibuprofen, indomethacin,ketoprofen, nabumetone, naproxen, sulindac, tenoxicam, tolmetin, andacetylsalicylic acid.

Suitable lipoxygenase inhibitors include redox inhibitors (e.g.,catechol butane derivatives, nordihydroguaiaretic acid (NDGA),masoprocol, phenidone, lanopalen, indazolinones, naphazatrom,benzofuranol, alkylhydroxylamine), and non-redox inhibitors (e.g.,hydroxythiazoles, methoxyalkylthiazoles, benzopyrans and derivativesthereof, methoxytetrahydropyran, boswellic acids and acetylatedderivatives of boswellic acids, and quinolinemethoxyphenylacetic acidssubstituted with cycloalkyl radicals), and precursors of redoxinhibitors.

Other suitable lipoxygenase inhibitors include antioxidants (e.g.,phenols, propyl gallate, flavonoids and/or naturally occurringsubstrates containing flavonoids, hydroxylated derivatives of theflavones, flavonol, dihydroquercetin, luteolin, galangin, orobol,derivatives of chalcone, 4,2′,4′-trihydroxychalcone, ortho-aminophenols,N-hydroxyureas, benzofuranols, ebselen and species that increase theactivity of the reducing selenoenzymes), iron chelating agents (e.g.,hydroxamic acids and derivatives thereof, N-hydroxyureas,2-benzyl-1-naphthol, catechols, hydroxylamines, carnosol trolox C,catechol, naphthol, sulfasalazine, zyleuton, 5-hydroxyanthranilic acidand 4-(omega-arylalkyl)phenylalkanoic acids), imidazole-containingcompounds (e.g., ketoconazole and itraconazole), phenothiazines, andbenzopyran derivatives.

Yet other suitable lipoxygenase inhibitors include inhibitors ofeicosanoids (e.g., octadecatetraenoic, eicosatetraenoic,docosapentaenoic, eicosahexaenoic and docosahexaenoic acids and estersthereof, PGE1 (prostaglandin E1), PGA2 (prostaglandin A2), viprostol,15-monohydroxyeicosatetraenoic, 15-monohydroxy-eicosatrienoic and15-monohydroxyeicosapentaenoic acids, and leukotrienes B5, C5 and D5),compounds interfering with calcium flows, phenothiazines,diphenylbutylamines, verapamil, fuscoside, curcumin, chlorogenic acid,caffeic acid, 5,8,11,14-eicosatetrayenoic acid (ETYA),hydroxyphenylretinamide, Ionapalen, esculin, diethylcarbamazine,phenantroline, baicalein, proxicromil, thioethers, diallyl sulfide anddi-(1-propenyl) sulfide.

Leukotriene receptor antagonists include calcitriol, ontazolast, BayerBay-x-1005, Ciba-Geigy CGS-25019C, ebselen, Leo Denmark ETH-615, LillyLY-293111, Ono ONO-4057, Terumo TMK-688, Boehringer Ingleheim BI-RM-270,Lilly LY 213024, Lilly LY 264086, Lilly LY 292728, Ono ONO LB457, Pfizer105696, Perdue Frederick PF 10042, Rhone-Poulenc Rorer RP 66153,SmithKline Beecham SB-201146, SmithKline Beecham SB-201993, SmithKlineBeecham SB-209247, Searle SC-53228, Sumitamo SM 15178, American HomeProducts Way 121006, Bayer Bay-o-8276, Warner-Lambert CI-987,Warner-Lambert CI-987BPC-15LY 223982, Lilly LY 233569, Lilly LY-255283,MacroNex MNX-160, Merck and Co. MK-591, Merck and Co. MK-886, OnoONO-LB-448, Purdue Frederick PF-5901, Rhone-Poulenc Rorer RG 14893,Rhone-Poulenc Rorer RP 66364, Rhone-Poulenc Rorer RP 69698, ShionoogiS-2474, Searle SC-41930, Searle SC-50505, Searle SC-51146, SearleSC-52798, SmithKline Beecham SKand F-104493, Leo Denmark SR-2566, TanabeT-757 and Teijin TEI-1338.

The invention as described in the examples is not intended to limit thescope of the invention. Cell lines described in the following exampleswere maintained in culture according to the conditions specified by theAmerican Type Culture Collection (ATCC) or Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH, Braunschweig, Germany (DMSZ), oras otherwise known. Cell culture reagents were obtained from InvitrogenCorp., Carlsbad, Calif. The present invention is not to be limited inscope by the specific embodiments described herein. Indeed, variousmodifications of the invention in addition to those described hereinwill become apparent to those skilled in the art from the foregoingdescription and accompanying figures. Such modifications are intended tofall within the scope of the appended claims.

What is claimed is:
 1. A compound comprising Formula (VIII) or (IX),wherein the compound is an αCD70 antibody conjugated to a dolastatin,wherein the conjugation occurs via a non-naturally encoded amino acid inthe antibody, wherein Formula (VIII) and (IX) correspond to:

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; B is optional, andwhen present is a linker selected from the group consisting of loweralkylene, substituted lower alkylene, lower alkenylene, substitutedlower alkenylene, lower heteroalkylene, substituted lowerheteroalkylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,—S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkyleneor substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substitutedalkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-,—C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene orsubstituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—,—C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—,where each R′ is independently H, alkyl, or substituted alkyl; R is H,alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R₁ isH, or an anti-CD70 (αCD70) antibody; R₂ is OH, or an anti-CD70 (αCD70)antibody; R₃ and R₄ are each independently H, halogen, lower alkyl, orsubstituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally form acycloalkyl or a heterocycloalkyl; Z has the structure of:

R₅ is H, COR₈, C₁-C₆alkyl, or thiazole; R₈ is OH; R₆ is OH or H; Ar isphenyl or pyridine; R₇ is C₁-C₆alkyl or hydrogen; L is a linker selectedfrom the group consisting of alkylene, -alkylene-C(O)—,-(alkylene-O)_(n)-alkylene-, -(alkylene-O)_(n)-alkylene-C(O)—,-(alkylene-O)_(n)—(CH₂)_(n′)—NHC(O)—(CH₂)_(n″)—C(Me)₂-S—S—(CH₂)_(n′″)—NHC(O)-(alkylene-O)_(n″″)-alkylene-,-(alkylene-O)_(n)-alkylene-W—, -alkylene-C(O)—W—,-(alkylene-O)_(n)-alkylene-U-alkylene-C(O)—, and-(alkylene-O)_(n)-alkylene-U-alkylene-; W has the structure of:

U has the structure of:

and each n, n′, n″, n′″ and n″″ are independently integers greater thanor equal to one; with the proviso that one and only one of R₁ or R₂ isan anti-CD70 (αCD70) antibody; or a pharmaceutically acceptable prodrugthereof.
 2. The compound of claim 1, wherein R₁ is H and R₂ is ananti-CD70 (αCD70) antibody.
 3. The compound of claim 1, wherein R₁ is ananti-CD70 (αCD70) antibody and R₂ is OH.
 4. The compound of claim 1,wherein the anti-CD70 (αCD70) antibody comprises no more than tennon-naturally encoded amino acids.